Factores inmunológicos asociados a la aparición de eventos no sida y al control espontáneo de la infección por el VIH.

La presente Tesis Doctoral se ha centrado en estudiar uno de los principales problemas clínicos de los pacientes infectados por VIH bajo tratamiento antirretroviral combinado (TARc), que es la prevalencia y morbi-mortalidad de procesos no definitorios de SIDA, entre los que se incluyen las enfermedades cardiovasculares. En un diseño longitudinal nosotros hemos observados que, elevados niveles de linfocitos T-CD8+ que expresan el marcador CCR5 junto a altos niveles de monocitos que expresan diferentes marcadores de activación, preceden a la aparición de eventos cardiovasculares en pacientes infectados por VIH bajo TARc supresor (Tarancon-Diez L et al. Throm Haemost. 2017). En este mismo escenario también hemos estudiado la asociación de un polimorfismo en el receptor 4 tipo-toll (TLR4) con el desarrollo de enfermedades cardiovasculares. Encontramos que la variante genética TLR4 Asp299Gly se asocia con eventos cardiovasculares, así como con un perfil proinflamatorio de los monocitos (Tarancon-Diez L et al. AIDS 2018). Además de la importancia del polimorfismo Asp299Gly, en este trabajo encontramos una asociación entre la coinfección por VHC e infecciones bacterianas con la aparición de enfermedades cardiovasculares. Estas observaciones fueron posteriormente corroboradas en una cohorte mayor, confirmando el papel de la coinfección por VHC junto con el efecto acumulativo de otras infecciones en el desarrollo de estas patologías (Genebat M, Tarancon-Diez L et al. Manuscrito en preparación). En relación con estos resultados, también hemos encontrado una asociación de la deleción delta32 en el gen CCR5 (CCR5Δ32) en heterocigosis, con una mejor progresión clínica en pacientes infectados por VIH durante la era de Tratamientos Antirretrovirales de Gran Actividad (TARGA) (Ruiz-Mateos E, Tarancon-Diez L et al. Antiviral Res. 2017). El escenario descrito hasta ahora se refiere a lo que ocurre en la mayoría de los pacientes. Sin embargo, existe una pequeña proporción (<1%) que tienen la peculiaridad de mantener espontáneamente la carga viral indetectable durante largos periodos de tiempo en ausencia de tratamiento antirretroviral; son los “controladores de élite” (CE). La segunda parte de la presente Tesis Doctoral se ha centrado en el fenómeno de control espontáneo del VIH. Hemos identificado diferencias inmunológicas y virológicas entre CE que a lo largo del tiempo, pierden el control virológico espontáneo del VIH y CE que mantienen persistentemente el control virológico; nuestros hallazgos permiten una definición fenotípica más exhaustiva del controlador, que puede constituir un modelo de “cura funcional”, con potencial impacto en el diseño de vacunas y estrategias inmunoterapéuticas. Adicionalmente en estos sujetos observamos un perfil proteómico y metabólico característico, bajo nivel de marcadores de inflamación, baja diversidad viral y alta polifuncionalidad anti-VIH específica (Rodriguez-Gallego E, Tarancon-Diez L et al. J Infect Dis. 2018, Tarancon-Diez L et al. Manuscrito en preparación)

altered expression of cD300a inhibitory receptor on cD4+ T cells From human immunodeficiency Virus-1-infected Patients: association With Disease Progression Markers.

The ability of the CD300a inhibitory receptor to modulate immune cell functions and its involvement in the pathogenesis of many diseases has aroused a great interest in this molecule. Within human CD4+ T lymphocytes from healthy donors, the inhibitory receptor CD300a is differentially expressed among different T helper subsets. However, there are no data about the expression and regulation of CD300a receptor on CD4+ T cells from human immunodeficiency virus (HIV)-1-infected patients. The objective of this study was to investigate the expression of CD300a on CD4+ T cells from HIV-infected patients on suppressive combined antiretroviral therapy (cART) and cART naïve patients. Our results have demonstrated that the expression levels of this inhibitory receptor were higher on CD4+ T cells from HIV-1 infected subjects compared with healthy donors, and that cART did not reverse the altered expression of CD300a receptor in these patients. We have observed an increase of CD300a expression on both PD1+CD4+ and CD38+CD4+ T cells from HIV-1 infected people. Interestingly, a triple positive (CD300a+PD1+CD38+) subset was expanded in naïve HIV-1 infected patients, while it was very rare in healthy donors and patients on cART. Finally, we found a negative correlation of CD300a expression on CD4+ T lymphocytes and some markers associated with HIV-1 disease progression. Thus, our results show that HIV-1 infection has an impact in the regulation of CD300a inhibitory receptor expression levels, and further studies will shed light into the role of this cell surface receptor in the pathogenesis of HIV infection

Immunometabolism is a key factor for the persistent spontaneous elite control of HIV-1 infection

Approximately 25% of elite controllers (ECs) lose their virological control by mechanisms that are only partially known. Recently, immunovirological and proteomic factors have been associated to the loss of spontaneous control. Our aim was to perform a metabolomic approach to identify the underlying mechanistic pathways and potential biomarkers associated with this loss of control. Methods: Plasma samples from EC who spontaneously lost virological control (Transient Controllers, TC, n=8), at two and one year before the loss of control, were comparedwith a control group of ECwho persistently maintained virological control during the same follow-up period (Persistent Controllers, PC, n=8). The determination of metabolites and plasma lipids was performed by GC-qTOF and LC-qTOF using targeted and untargeted approaches. Metabolite levels were associated with the polyfunctionality of HIV-specific CD8+T-cell response. Findings: Our data suggest that, before the loss of control, TCs showed a specific circulating metabolomic profile characterized by aerobic glycolytic metabolism, deregulated mitochondrial function, oxidative stress and increased immunological activation. In addition, CD8+ T-cell polyfunctionality was strongly associated with metabolite levels. Finally, valine was the main differentiating factor between TCs and PCs. Interpretation: All these metabolomic differences should be considered not only as potential biomarkers but also as therapeutic targets in HIV infection.Instituto Carlos III PI10/02635 PI13/00796 PI16/00503 PI12/02283 PI16/00684 CPII014/00025 FI14/00431 FI17/00186 INT11/240 INT12/282 INT15/226Fondos Europeos para el Desarrollo Regional PI10/02635 PI13/00796 PI16/00503 PI12/02283 PI16/00684 CPII014/00025 FI14/00431 FI17/00186FEDER PI10/02635 PI13/00796 PI16/00503 PI12/02283 PI16/00684 CPII014/00025 FI14/00431 FI17/00186FEDER PI10/02635 PI13/00796 PI16/00503 PI12/02283 PI16/00684 CPII014/00025 FI14/00431 FI17/00186Programa de Suport als Grups de Recerca 2017SGR948 2014SGR250Gilead Fellowship Program GLD14/293 GLD17/00299Red de Investigación en Sida RD12/0017/0005 RD16/0025/0006 RD12/0017/0029 RD16/0025/0020Junta de Andalucía C-0032/17Generalitat de Catalunya PERIS SLT002/16/0010

Immune Correlates of Natural HIV Elite Control and Simultaneous HCV Clearance—Supercontrollers

HIV-elite controllers are a minority group of HIV-infected patients with the ability to maintain undetectable HIV viremia for long time periods without antiretroviral treatment. A small group of HIV-controllers are also able to spontaneously clear the hepatitis C virus (HCV) whom we can refer to as “supercontrollers.” There are no studies that explore immune correlates looking for the mechanisms implicated in this extraordinary phenomenon. Herein, we have analyzed HCV- and HIV-specific T-cell responses, as well as T, dendritic and NK cell phenotypes. The higher HCV-specific CD4 T-cell polyfunctionality, together with a low activation and exhaustion T-cell phenotype was found in supercontrollers. In addition, the frequency of CD8 CD161high T-cells was related with HIV- and HCV-specific T-cells polyfunctionality. Interesting features regarding NK and plasmacytoid dendritic cells (pDCs) were found. The study of the supercontroller's immune response, subjects that spontaneously controls both chronic viral infections, could provide further insights into virus-specific responses needed to develop immunotherapeutic strategies in the setting of HIV cure or HCV vaccination

Immunoescape of HIV-1 in Env-EL9 CD8 + T cell response restricted by HLA-B*14:02 in a Non progressor who lost twenty-seven years of HIV-1 control

Background: Long-Term Non-Progressors (LTNPs) are untreated Human Immunodeficiency virus type 1 (HIV-1) infected individuals able to control disease progression for prolonged periods. However, the LTNPs status is temporary, as viral load increases followed by decreases in CD4 + T-cell counts. Control of HIV-1 infection in LTNPs viremic controllers, have been associated with effective immunodominant HIV-1 Gag-CD8 + T-cell responses restricted by protective HLA-B alleles. Individuals carrying HLA-B*14:02 control HIV-1 infection is related to an immunodominant Env-CD8 + T-cell response. Limited data are available on the contribution of HLA-B*14:02 CD8 + T -cells in LTNPs. Results: In this study, we performed a virological and immunological detailed analysis of an HLA-B*14:02 LNTP individual that lost viral control (LVC) 27 years after HIV-1 diagnosis. We analysed viral evolution and immune escape in HLA-B*14:02 restricted CD8 + T -cell epitopes and identified viral evolution at the Env-EL9 epitope selecting the L592R mutation. By IFN-γ ELISpot and immune phenotype, we characterized HLA- B*14:02 HIV-1 CD8 + T cell responses targeting, Gag-DA9 and Env-EL9 epitopes before and after LVC. We observed an immunodominant response against the Env-EL9 epitope and a decreased of the CD8 T + cell response over time with LVC. Loss of Env-EL9 responses was concomitant with selecting K588R + L592R mutations at Env-EL9. Finally, we evaluated the impact of Env-EL9 escape mutations on HIV-1 infectivity and Env protein structure. The K588R + L592R escape variant was directly related to HIV-1 increase replicative capacity and stability of Env at the LVC. Conclusions: These findings support the contribution of immunodominant Env-EL9 CD8 + T-cell responses and the imposition of immune escape variants with higher replicative capacity associated with LVC in this LNTP. These data highlight the importance of Env-EL9 specific-CD8 + T-cell responses restricted by the HLA-B*14:02 and brings new insights into understanding long-term HIV-1 control mediated by Env mediated CD8 + T-cell responses.Molecular Virology Laboratory was supported by grants SAF (2016-77894-R) from Ministerio de Economía y Competitividad (MINECO), ISCIII through the projects PI 13/02269, PI17/00164, PI16/0684, PI19/01127 (Co-funded by European Regional Development Fund/European Social Fund "Investing in your future"). The RIS-RETIC grants RD12/0017/0028, RD16/0025/0020 and RD16CIII/0002/0005. LTD was supported by the Instituto de Salud Carlos III (ISCIII) under grant agreement “CD20/00025” through the Sara Borrell Program. O.B.L was funded by an AGAUR-FI_B 00582 Ph.D. fellowship from the Catalan Government and the European Social Fund. M.A. was funded by grants RYC-2015-18241 and PID2019-107931GA-I00 from the Spanish Government and, ED431F 2018/08 from the “Xunta de Galicia”. ERM was supported by the Spanish National Research Council (CSIC). JGP laboratory was supported by National Health Institute Carlos III grant PI17/00164 and Redes Temáticas de Investigación en SIDA (ISCIII RETIC RD16/0025/0041). The funders had no role in study design, data collection and analysis, the decision to publish or drafting of the manuscript.S

Permanent control of HIV-1 pathogenesis in exceptional elite controllers: a model of spontaneous cure

Elite controllers (EC) represent a small subset of HIV-1-infected people that spontaneously control viral replication. However, natural virological suppression and absence of immune dysfunction are not always long-term sustained. We define exceptional EC (EEC) as HIV-1 subjects who maintain the EC characteristics without disease progression for more than 25 years. We analyzed three EEC, diagnosed between 1988 and 1992, who never showed signs of clinical disease progression in absence of any antiretroviral treatment. A comprehensive clinical, virological, and immunological study was performed. The individuals simultaneously exhibited ≥3 described host protective alleles, low levels of total HIV-1 DNA (0.50). Inflammation levels of EEC were similar to HIV-1 negative donors. Remarkably, they showed an exceptional lack of viral evolution and 8-fold lower genetic diversity (<0.01 s/n) in env gene than other EC. We postulate that these EEC represent cases of spontaneous functional HIV-1 cure. A non-functional and non-genetically evolving viral reservoir along with an HIV-1-specific immune response seems to be key for the spontaneous functional cure.Work in Centro Nacional de Microbiologia (ISCIII) was supported by grants SAF (2016–77894-R) from Ministerio de Economia y Competitividad (MINECO) (Spain) and Fondo de Investigación Sanitaria (FIS)-Instituto de Salud CarlosIII, grant FIS (PI 13/02269, ISCIII) and in part by the RIS-RETIC grants RD12/0017/0028 and RD16CIII/0002/0005 funded by the ISCIII-FEDER. MP has a contract of RIS-RETIC RD16CIII/0002/0005. This work was supported by grants from the MINECO, FIS-Instituto de Salud CarlosIII, Fondos Europeos para el Desarrollo Regional, FEDER, grant numbers PI16/00684, PI19/01127, CPII014/00025 to ER-M. and FI14/00431 to LT-D.; the Gilead Fellowship Program (grant numbers GLD17/00299); the Red de Investigación en Sida (grant number RD16/0025/0020). ER-M. is supported by Consejería de Salud y Bienestar Social of Junta de Andalucía through the Nicolás Monardes Program (C-0032/17). Research in VS-M group was supported by Fondo de Investigación Sanitaria (FIS)-Instituto de Salud CarlosIII, grant FIS (PI 17CIII/00049). Grifols partially supported work in the AIDS Research Institute IrsiCaixa. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Persistent HIV-controllers are more prone to spontaneously clear HCV: a retrospective cohort study.

HIV-controllers have the ability to spontaneously maintain viraemia at low or undetectable levels in the absence of antiretroviral treatment. Furthermore, HIV-controllers seem to have a superior capacity to spontaneously clear hepatitis C virus (HCV) compared to non HIV-controllers. Some of these subjects eventually lose HIV-controller status (transient controllers), whereas some HIV-controllers show a persistent natural HIV control (persistent controllers). We aimed to analyse whether persistent controllers have superior capacity to spontaneously clear HCV compared to transient controllers. We recruited HIV-controllers from January 1981 up to October 2016 with available antibodies to HCV (anti-HCV) data (n = 744). Factors associated with HIV spontaneous control in relation to HCV status were analysed in persistent and transient HIV-controllers with anti-HCV positive (n = 202 and n = 138 respectively) in comparison with 1700 HCV positive non HIV-controllers recruited from January 1981 up to March 2018, bivariate and multivariate analyses, following a logistic regression model, were applied. In addition, the factors related to the loss and time to lose HIV-controller status were explored (n = 744) using Log rank test and Kaplan-Meier curves, in this case the multivariate analysis consisted in a Cox regression model. A higher frequency of HCV spontaneous clearance was found in persistent HIV-controllers (25.5%) compared to non-controllers (10.2%). After adjusting for potential confounders, as sex, age, HIV transmission risk, CD4+ T-cell nadir and time of follow-up, HCV clearance was independently associated with persistent HIV spontaneous control (p = 0.002; OR (95% CI) = 2.573 (1.428 to 4.633)), but not with transient spontaneous control (p = 0.119; 1.589 (0.888 to 2.845)). Furthermore, persistent HIV-controllers were more likely to spontaneously clear the HCV in comparison with transient controllers (p = 0.027; 0.377 (0.159 to 0.893). Finally, not to lose or lengthen the time of losing this control was independently associated with HCV spontaneous clearance (p = 0.010; 0.503 (0.297 to 0.850). This study shows an association between spontaneous persistent HIV-control and HCV spontaneous clearance. The study findings support the idea of preserved immune mechanisms in persistent HIV control implicated in HCV spontaneous clearance. These results highlight persistent HIV-controllers but not transient controllers as a good model of functional HIV cure.This work was supported by the Instituto de Salud Carlos III (research contracts CPII014/00025 to E.R.‐M., and FI14/00431 to L.T.‐D. and research projects PI12/02283, PI16/00684, PI19/01127 to E.R.‐M.) and Red Temática de Investigación Cooperativa en SIDA (Projects RD12/0017/0029, RD12/0017/0031, and RD16/0025/0020 and RD16/0025/0013), which is included in the Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008 to 2011 and 2013 to 2016, Instituto de Salud Carlos III, Fondos FEDER. E.R.‐M. was supported by Consejería de Salud y Bienestar Social of Junta de Andalucía through the Nicolás Monardes program (C‐0032/17), N Rallón is a Miguel Servet investigator from the Spanish Carlos III Institute of Health (ISCIII), grant CP14/00198, Madrid, Spain and B.D.M. received a grant from The Spanish Ministry of Education (FPU13/02451). Work in CL‐G’s laboratory was supported by grants SAF (2010 to 17226) and (2016‐77894‐R) from MINECO (Spain) and FIS (PI 13/02269, ISCIII) and in part by the RIS‐RETIC grants RD06/006/0036 and RD12/0017/0028 funded by the ISC III‐FEDER. MP has a contract of RIS‐RETIC RD12/0017/0036.S

Coordinate-based co-localization-mediated analysis of arrestin clustering upon stimulation of the C-C chemokine receptor 5 with RANTES/CCL5 analogues

G protein-coupled receptor activation and desensitization leads to recruitment of arrestin proteins from cytosolic pools to the cell membrane where they form clusters difficult to characterize due to their small size and further mediate receptor internalization. We quantitatively investigated clustering of arrestin 3 induced by potent anti-HIV analogues of the chemokine RANTES after stimulation of the C-C chemokine receptor 5 using single-molecule localization-based super-resolution microscopy. We determined arrestin 3 cluster sizes and relative fractions of arrestin 3 molecules in each cluster through image-based analysis of the localization data by adapting a method originally developed for co-localization analysis from molecular coordinates. We found that only classical agonists in the set of tested ligands were able to efficiently recruit arrestin 3 to clusters mostly larger than 150 nm in size and compare our results with existing data on arrestin 2 clustering induced by the same chemokine analogues