Phd in MOLECULAR MEDICINE

Thesis title: HIV-1 INFECTION: ROLE OF ANTIRETROVIRAL DRUGS AND CELL-ASSOCIATED MICRORNA IN THE CONTROL OF VIRAL REPLICATION

To date, HIV infection still represents a serious threat among infectious diseases, with over 38 million people infected in the world. The natural progression of the infection has changed since the introduction of combined antiretroviral therapy (cART) in the mid-90s, which leads to a substantial decrease in mortality and morbidity among the HIV positive population. However, treated patients have a shorter life expectancy than the seronegative counterpart due to the premature onset of non-AIDS related events, such as cardiovascular disease, neurocognitive disorders, bone abnormalities, metabolic syndrome, and non-HIV related malignancies. The cART long-term adverse effects and the non-normalization of inflammatory and immune system activation markers, even in patients treated with effective cART, have been reported to plays a pivotal role in the pathogenesis of these illnesses. Therefore, more efforts are needed to evaluate the potential of new antiretroviral strategies, single tablet and drug-sparing approaches, as maintenance regimens in order to improve the outcomes of long-term treated patients. In this setting, we first set out to investigate the efficacy, safety, and tolerability of switching to a dolutegravir (DTG)-based regimen in a cohort of individuals with HIV-1 infection with a stable virologic control. Besides, the dynamics of total HIV DNA and levels of high-sensitivity c-reactive protein, interleukin-6, soluble-CD14 and D-Dimer were analyzed. Of the 96 patients in the study, ninety-three maintained virological suppression, including patients who switched to dual-therapy from triple-drug combination. Overall, 18 patients had residual viremia at baseline, of which 13 reached the maximal viral suppression at W48. Serum creatinine level showed a significant increase at weeks 24 and 48 but without exceed reference values. In addition, a progressive reduction of total cholesterol was observed from week 24 and up to week 48. No variation in Body Mass Index was detected. HIV DNA, inflammation, and coagulation marker levels did not significantly change during follow-up. These results suggest that switching to a DTG-based regimen may be a key option for achieving and maintaining maximal virological suppression, even in patients showing residual viremia at baseline. Furthermore, the improvement in blood lipid profile and the overall tolerability observed firmly support the use of drug regimens with dolutegravir in the aging HIV population. As reported above, despite the improved efficacy and tolerability of new antiretroviral compounds, several patients did not achieve complete virological suppression showing residual levels of viral replication over time, known as Residual Viremia. The source of residual viremia has not been well established and most likely results from multiple mechanisms, such as ongoing rounds of viral replication in sanctuary sites and activation of infected cells in the latent viral reservoir. Recent findings have shown that factors closely related to the host could be associated with disease control. Particularly, cellular microRNAs, such as miR-150, miR-223, miR-382, have been shown to promote HIV-1 latency through the inhibition of viral proteins (Tat and Rev) translation in resting CD4+ T cells. Furthermore, reports have found the ability of miR-324-5p and miR33a-5p to hinder HIV-1 replication acting at different stages of its life cycle. On the other hand, it has been demonstrated that molecules of the same class of small non-coding RNA, such as miR-34a, miR-132, could enhance viral replication. Considering these evidence, we set out to investigate if miR-150, miR-223, miR-382, miR-324-5p, miR-33a-5p, miR-34a, miR-132 could be responsible for the different control of viral replication observed in treated patients. Levels of PBMC-associated miRNAs were analyzed in samples from 60 ART-treated patients stratified into two groups according to the viral load over the past three years: i) 30 patients with undetectable viremia (TND) ii) 30 patients with detectable low-level viremia (LLV, 1-200 copies/mL). In addition, 30 treatment-naïve patients were analyzed. The results were compared to those of the control group which includes 28 healthy donors (HD). The expression of miRNAs under examination shown to be affected by HIV and stratifying the infected population according to the viral load, distinct expression patterns have been observed. MiR-223 was differentially expressed among HIV+ groups with higher levels reported in TND patients. Since miR-223 exert an anti-HIV replication activity by targeting 3’ UTR sequences of HIV RNA in primary resting CD4+ cells and interfering with cyclin T1 expression, our results may provide new details into the host processes required to reach undetectable viremia. The lack of correlation with CD4+ T cell count and viral load, and the influence of the duration of HIV infection on the expression levels of some miRNAs, point to the involvement of other processes that could be related to the inflammatory and immune systems activation and/or to indirect effects of viral products. These processes could lead to an evolution of miRNAs response not confined to a particular cell subset. Therefore, further studies focused on CD4+ T cells and monocytes/macrophages lineages are needed to clarify their contribution to the miRNAs expression during HIV infection. Finally, patients’ stratification for therapeutic regimen showed a lower expression of miR-33a-5p in patients treated with non-nucleoside reverse transcriptase inhibitors (NNRTI) compared to those receiving protease (PI) or integrase inhibitors (INSTI). Although further investigations are necessary, this finding suggests a possible non-specific action of PI and INSTI in countering HIV-1 replication. In conclusion, these results support that the control of HIV replication is a complex process not confined to the viral characteristics and the efficacy of antiviral compounds, but also host-related factors, like miRNAs, could play a pivotal role in achieving and maintaining complete virological suppression. A further characterization of miRNAs profile in the different PBMCs cellular subsets and their circulating form could clarify their role during HIV disease and might provide new prognostic and treatment-response biomarkers useful to improve the management and the outcome of subjects with HIV infection.