In search of predictive biomarkers of the replication-competent HIV-1 reservoir

Quantification and exploration of replication-competent HIV-1 reservoirs in patients on stable suppressive treatment remains a major challenge for HIV-1 cure studies. The aim of my doctoral research was to gain insight in the size and dynamics of the HIV-1 reservoir and identify its most predictive biomarkers to guide HIV-1 cure studies. To address this goal, four research objectives were set, and patient samples from several well-defined cohorts were used. The first research objective was to identify and validate an HIV-1 biomarker representative of the latent reservoir, which can potentially be used in future monitoring of the effect of clinical trials aiming to purge the latent reservoir. For this purpose we designed, optimized, validated and subsequently compared assays to quantify cell-associated HIV-1 RNA using the digital (d)PCR to real-time (q)PCR, showing non-inferiority to the qPCR assay. The second research objective was to further validate HIV-1 persistence biomarkers, in a specifically selected patient cohort (N=161) that consisted of two different treatment arms, i.e. a non-nucleoside reverse transcriptase inhibitor based or a protease inhibitor based treatment regimen. These treatment regimens differ in their half-life. A broad range of HIV-1 reservoir biomarkers were evaluated: cell-associated unspliced and multiply spliced HIV-1 RNA, total HIV-1 DNA, and 2LTR circular DNA. No difference in reservoir size was observed between different high potent ART regiments. On the basis of the findings in long-term ART treated patients, an additional longitudinal analysis of HIV-1 biomarkers was performed using preselected samples of the previous study (N=25) to compare HIV-1 biomarkers with PCR based and cell-culture based assays to find a HIV-1 biomarker predictive of the latent reservoir. The study revealed important correlations between the viral outgrowth (cell-culture based) and total and integrated HIV-1 DNA (PCR based), hence, showing that the total pool of HIV-1 DNA is associated with the amount of replication-competent virus in patients on treatment. To further evaluate the mechanism of HIV-1 persistence, HIV-1 integration sites were analysed. For this last research goal, patients with either no inducible or very high amounts of inducible virus were selected. A small patient cohort was formed, comprising of chronically infected long-time ART treated HIV-1 infected patients. The integration site analysis was coupled with an estimate of the frequency of CD4+ T cells with inducible HIV-1 RNA transcription levels. The study showed that most of the ART treated patients in this study had an HIV-1 DNA reservoir consisting of 30% clonally expanded cells. Also, patients with higher clonal diversity, i.e. higher number of clones of proliferated cells detected, had higher estimate for inducible HIV-1 RNA transcription. The work done for this PhD thesis contributes to the knowledge of HIV-1 latent reservoirs by exploring HIV-1 biomarkers in several patient cohorts. We designed an assay for assessment of cell-associated HIV-1 RNA using dPCR and we have shown that different potent ART compositions do not affect the size of the latent reservoir. We showed important correlations between PCR and cell-culture based assays for HIV-1 reservoir quantification. These results suggest that total HIV-1 DNA could be used as a biomarker to predict the size of the replication-competent latent HIV-1 reservoir. Finally, we added new knowledge on the frequency of clonally expanded cells in patients on long-term ART