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Title: HIV-1 persistence during ART
Author: Ruggiero, Alessandra
ISNI:       0000 0004 6059 5085
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Antiretroviral therapy (ART) suppresses HIV viremia, but it does not eliminate the stable cellular reservoir of integrated viral DNA that is generated early during infection. As a consequence, low levels of HIV are detected in patients with consistent viral suppression (HIV-1 RNA < 50 copies/ml) in CD4 cells in different body compartments including plasma, peripheral cells and CD4 T-cell subsets. We first explored the levels of HIV-1 RNA below the cut-off of clinical assay (HIV-1 RNA load < 50 copies/ml, herein referred to as 'residual viremia') in a group of patients who switched from Atripla to Eviplera due to toxicity. Furthermore, we studied HIV-1 DNA and RNA loads in peripheral blood of patients with stable virological suppression (HIV-1 RNA < 50 copies/ml) while being on first line-ART with 1 NNRTI and 2 NRTIs. The measured virological markers included the residual HIV-1 RNA load in plasma quantified by an ultrasensitive assay; total and integrated HIV-1 DNA in both peripheral blood mononuclear cells (PBMC) and the CD4 T-cell subsets using quantitative PCR methods; 2-LTR circular HIV-1 DNA in PBMC using droplet digital PCR assay. Furthermore, a panel of cellular and immunological markers of immune activation, as measured by flow cytometry and ELISA assay respectively, were available for the analysis. Additionally we explored alternative methods to quantify and to study HIV-1 persistence. In patients that switched from Atripla to Eviplera we observed that overall the patients showed maintained virologic suppression until the end of the observation; however, 4 patients experienced virologic rebound with HIV-1 RNA > 50 copies/ml and this was predicted by a progressive increase in residual HIV-1 RNA levels over time. Patients on suppressive ART were studied with a cross sectional approach and we observed detection of residual HIV-1 RNA and 2-LTR circular DNA in a proportion of patients and total HIV-1 DNA in PBMC the whole study group. Additionally, we found that residual HIV-1 RNA levels and total HIV-1 DNA were associated with sCD27 and CD8+HLA-DR/DP/DQ+ T-cells, respectively. Following setting-up of the Alu-gag qPCR assay, we measured integrated HIV-1 DNA in PBMC and we confirmed the association with CD8+HLA-DR/DP/DQ+ T-cells. In the context of assay development, we proposed the Alu-5LTR assay as an alternative to the Alu-gag assay for integrated HIV-1 DNA quantification and preliminary results showed increased sensitivity. Furthermore, we identified and characterized the tools needed to set up an in vitro system to study direct and cell-to-cell HIV-1 infection. Results from my thesis showed the importance of residual HIV-1 RNA monitoring to detect virus rebound. Moreover, patients on suppressive ART for a long period showed HIV detection in plasma and in cells which was associated with high levels of immune activation, suggesting a role for the host immune environment in HIV persistence. In the context of assay developments for more sensitive quantification of integrated HIV DNA reservoir, our proposed Alu-5LTR assay as described in this thesis may be a potential alternative.
Supervisor: Geretti, A. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral