Ruiz- Mateos Carmona, EzequielNeukam, KarinGasca-Capote, Carmen2025-06-042025-06-042025-02-07Gasca-Capote, C. (2025). Persistent Controllers as the key Model to identify permanent HIV Remission. (Tesis Doctoral Inédita). Universidad de Sevilla, Sevilla.https://hdl.handle.net/11441/173928Elite controllers (ECs) are a heterogeneous and dynamic group regarding virological, immunological and clinical factors. Approximately 25% of ECs eventually loss virological control. This has enabled to classify ECs in two different phenotypes: persistent elite controllers (PCs), people with HIV (PWHIV) that permanently maintain virological control in the absence of antiretroviral treatment (ART); and transient elite controllers (TCs), PWHIV who eventually lost the virological control after being able to control viral replication without ART. Several studies have shown that PCs and TCs present different immunological, virological, proteomic, metabolomic and microRNA (miRNA) profiles. However, in terms of virological factors, it is essential to deeply characterize the quality of the human immunodeficiency virus (HIV) reservoirs to distinguish both phenotypes. This differentiation is key to identifying the factors that lead to HIV progression and could pave the way for new strategies in the pursuit of an HIV cure. Seventeen PCs, that have maintained virological control for a median of 25.5 [22.3-31.3] years; ten TCs with sustained viral load above the detection limit, >40 HIV RNA copies/mL, during more than one year of follow-up before losing the virological control for a median of 1.2 [0.5-1.7] years; and 41 PWHIV on ART, were included in the study. The characterization of the HIV-1 reservoir, from peripheral blood mononuclear cells (PBMCs), was performed using next-generation sequencing (NGS) techniques, such as full-length individual proviral sequencing (FLIP-seq), matched integration site and proviral sequencing (MIP-seq) and integration site loop amplification (ISLA). The immune footprints to broadly neutralizing antibodies (bnAbs) and to cytotoxic T-lymphocyte (CTL) escape mutations were determined by intact and defective proviral sequences. The cell-associated HIV-1 RNA levels and the thymic function were quantified by droplet digital PCR (ddPCR), from previously extracted RNA and DNA, respectively. The CD8+ T cell proliferation and HIV-specific T cell response assays were performed by multiparametric flow cytometry after stimulation with gag peptides. PCs and TCs before losing the virological control, presented significantly lower total, intact and defective proviruses compared to participants on ART. Although no significant difference was found in total and defective proviruses between PCs and TCs, the proportion of intact proviruses were significantly lower in PCs compared to TCs; indeed, the intact/defective HIV-DNA ratio was significantly higher in TCs. Moreover, non-clonally expanded intact proviruses were found in TCs, indicating a greater viral diversity in comparison to PCs. Interestingly, no genome-intact HIV-1 was detected in most of the PCs, predominantly among females. Furthermore, intact proviruses from TCs were located into permissive genic euchromatic positions and presented higher resistance to bnAbs recognition, before losing virological control, in contrast to PCs whose intact proviruses were located in centromeric satellite DNA or zinc-finger genes, both associated with heterochromatin features. Cell-associated HIV-1 RNA levels were significantly higher in TCs, and not detected in five PCs that corresponded to the participants with no intact provirus detected. Lower levels of Gag-specific T cell response polyfunctionality and higher CD8+ T cell proliferation and thymic function were found in TCs before losing virological control compared to PCs. Our findings revealed a markedly distinct intact and defective proviral reservoir, and immunological landscapes associated with the loss and maintenance of persistent spontaneous HIV-control. These results suggest the need for, and can give guidance to, the design of future research to identify a distinct proviral landscape that may be associated with the persistent control of HIV-1 without ART.application/pdf148 p.engAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccess