Browsing by Author "Ghiglione, Y"

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    Bioinformatic analysis of post-transmission viral readaptation in Argentine patients with acute HIV-1 infection
    (2020-07) Damilano, G; Sued, Omar; Satorres, S; Ruiz, Maria; Ghiglione, Y; Guzman, F; Turk, Gabriela ; Quiroga, F; Cahn, Pedro; Salomon, Horacio; Dilernia, Dario
    During the acute phase of HIV-1 infection, a strong readaptation occurs in the viral population. Our objective was to analyze the post-transmission mutations associated with escape to the cytotoxic immune response and its relationship with the progression of the infection. In this study, a total of 17 patients were enrolled during acute/early primary HIV infection and 8 subjects that were the HIV positive partner resulting in 8 transmission pairs. Genotyping of the genetic polymorphisms of HLA class I A and B was performed using PCR-SSOP. Viral RNA extraction was from plasma. 570 single Gag-gene amplifications were obtained by limiting-dilution RT-PCR. Epitope prediction was performed with NetMHC CBS prediction server for the 19 HLA-A and single bondB alleles. Cytotoxic response prediction was performed by using the IEDB Analysis Resource. From our results, we deduce that the transmitted CTL / gag escape frequency in the founder virus was at least double compared to the post-transmission events. Additionally, by means of an algorithm that combines these frequencies, we observed that the founder viruses better adapted to the HLA A / B alleles of the recipient could contribute to a greater progression of the infection. Our results suggest that there is a large adaptation of HIV-1 to the HLA A / B alleles prevalent in our population. However, despite this adaptive advantage, the virus needs to make “readjustments” through new escape and compensatory mutations. Interestingly, according to our results, this readaptation could have a role in the progression of the infection.
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    Functionality of CD8+ T-cells in subjects under cART: implications on cure strategies
    (2018-08) Salido, J; Ruiz, Maria; Trifone, C; Figueroa, MI; Caruso, MP; Gherardi, MM; Sued, Omar; Horacio, S; Natalia, L; Ghiglione, Y; Turk, Gabriela
    Background: Reaching HIV cure will largely depend on the capacity of HIV-specific memory CD8+ T-cells (CD8TC) to eliminate the viral reservoir. However, CD8TC response is limited in subjects on cART. Here, we aimed to investigate the phenotype and function of in vitro expanded CD8TCs in HIV+ subjects and the impact of ART initiation timing on these parameters. Methods & Materials: PBMCs from 28 HIV+ subjects on cART for 1 year were obtained. Twelve initiated treatment during chronic infection (Delayed Treatment, DT) and 16 within four months post-infection (Early Treatment, ET). PBMCs were stimulated with peptides spanning Nef and Gag plus IL-2 during 14 days. ELISPOT (pre and post-expansion) and Flow Cytometry (FC, post-expansion) were performed to assess expanded CD8TC function (CD107a/b, IFN-g, IL-2, MIP-1b and TNF-a) and phenotype (CD45RO, CCR7, CD95 and PD1). Data was analyzed using non-parametric statistics. Results: Magnitude of ELISPOT responses increased after expansion by 103 times (p < 0.002), in both groups, being this effect more pronounced in CD8TCs, compared to CD4TCs (p < 0.0001), as confirmed by FC. Cells showed higher avidity after stimulation (evidenced by greater spot sizes, p < 0.002). DT subjects displayed a broader response to HIV than ET, after expansion. ET group had a significantly higher proportion of monofunctional degranulating CD8TCs (CD107a/b+), when challenged against Gag peptides (p = 0.037) compared to DT. Contrary, DT group showed higher polyfunctionality (p = 0.009). In both groups, CD4TC responses were of lesser magnitude compared to CD8TCs and predominantly monofunctional. Bulk and HIV-specific CD8TC phenotype varied significantly between groups: ET subjects showed a preservation of stem and central memory cells while DT showed a fully-differentiated profile (p < 0.005). When analyzing memory distribution within PD1+CD8+ cells, terminal effector were the most frequent subpopulation in DT and effector memory cells in ET individuals; evidencing a differential cell exhaustion profile in both groups. Conclusion: We demonstrated that HIV-specific CD8TCs could be selectively stimulated and expanded in subjects under ART. We also showed that ART initiation timing has an impact on phenotype and function of CD8TCs, reflecting consequences of longer antigen persistence on immune function. Overall, results presented in this work have important implications for the development of cure strategies aim at boosting CD8TC responses.
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    Phenotype and functionality of CD8+ T cells before and after cART are related to the viral reservoir size in people living with HIV-1
    (2019) Czernikier, A; Salido, J; Trifone, C; Figueroa, MI; Salomon, Horacio; Cahn, Pedro; Sued, Omar; Laufer, N; Ghiglione, Y; Trifone, C
    Background: the persistence of latently infected t-cells remains the major obstacle to cure HIV. special emphasis has been placed to identify the characteristics of cD8+ t-cells (cD8tcs) associated with viral control. We aimed to determine the relationship between the quality of the immune response before and after antiretroviral treatment (cart) and HIV persistence on cart. Methods: 18 subjects were enrolled during acute/early HIV infection (median 2 month post-infection). blood samples were obtained at enrollment (baseline sample, pre-cart) and after 18 months post-art (on-cart). Phenotypic (cD45ro, ccr7, cD95, PD-1) and functional (cD107, cytokines) markers were studied on bulk and HIV-specific cD8tcs by flow cytometry, in both samples. cell- associated HIV DNa forms (total and integrated) were quantitated by real-time Pcr in samples on-cart. Data was analyzed using non-parametric statistics. Results: spearman’s correlations showed that higher HIV-integrated DNa levels on samples on-cart were related to lower expression of baseline cD107+ cD8tcs and lower proportion of HIV-specific terminal effector (te) cD8tcs (p=0,003 and p=0,049). Moreover, total HIV DNa levels post-cart directly correlated with baseline HIV-specific PD-1+cD8tcs. HIV-integrated DNa levels positively correlated with the percent- age of PD-1+cD8tcs (p< 0,0001), and inversely with the per- centage of bi- and trifunctional cD8tcs (p=0,024 and p=0,048) at on-cart samples. Moreover, direct correlations between the percentage of effector memory cD8tcs at: bulk, HIV-specific, and PD-1+ compartments and the levels of HIV-integrated DNa were observed (p= 0,005, p=0,0003 and p=0,004). similarly, HIV-integrated DNa inversely correlated with the proportion of cD8tc te in the same three compartments (p=0,016, p=0,0003 and p=0,007). Finally, negative correlations between HIV- integrated DNa levels and the percentage of naïve and stem cell memory cD8tcs (r=-0,561, p=0,002; r=-0,398, p=0,044) were found. Conclusions: Different immune parameters evaluated pre- and post-cart were related to HIV persistence. overall, results suggest that an exhausted and dysfunctional HIV-specific immune response, both before and after treatment initiation, was related to a higher reservoir size at 18 months post-cart. this should be considered when designing functional cure approaches. also, the potential use of these parameters as markers of reservoir size and/or remission should be studied.