Regulacion de la expresion del VIH-1 por la modulacion de NF?B/I?Bα: modificaciones celulares inducidas por la proteina Tat del VIH-1

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-10-200

Molecular mechanisms involved in HIV latency and implications for HIV treatment and eradication

The aim of this presentation is to review the molecular mechanisms necessary for the establishment of HIV-1 latency, their relationship with different cellular and anatomic reservoirs, as well as the current treatment strategies targeting viral persistence in latent reservoirs, their main limitations and future perspectives.S

Transcriptomic Evidence of the Immune Response Activation in Individuals With Limb Girdle Muscular Dystrophy Dominant 2 (LGMDD2) Contributes to Resistance to HIV-1 Infection

LGMDD2 is a rare form of muscular dystrophy characterized by one of the three heterozygous deletions described within the TNPO3 gene that result in the addition of a 15-amino acid tail in the C-terminus.TNPO3 is involved in the nuclear import of splicing factors and acts as a host cofactor for HIV-1 infection by mechanisms not yet deciphered. Further characterization of the crosstalk between HIV-1 infection and LGMDD2 disease may contribute to a better understanding of both the cellular alterations occurring in LGMDD2 patients and the role of TNPO3 in the HIV-1 cycle. To this regard, transcriptome profiling of PBMCs from LGMDD2 patients carrying the deletion c.2771delA in the TNPO3 gene was compared to healthy controls. A total of 545 differentially expressed genes were detected between LGMDD2 patients and healthy controls, with a high representation of G protein-coupled receptor binding chemokines and metallopeptidases among the most upregulated genes in LGMDD2 patients. Plasma levels of IFN-β and IFN-γ were 4.7- and 2.7-fold higher in LGMDD2 patients, respectively. An increase of 2.3-fold in the expression of the interferon-stimulated gene MxA was observed in activated PBMCs from LGMDD2 patients after ex vivo HIV-1 pseudovirus infection. Thus, the analysis suggests a pro-inflammatory state in LGMDD2 patients also described for other muscular dystrophies, that is characterized by the alteration of IL-17 signaling pathway and the consequent increase of metallopeptidases activity and TNF response. In summary, the increase in interferons and inflammatory mediators suggests an antiviral environment and resistance to HIV-1 infection but that could also impair muscular function in LGMDD2 patients, worsening disease evolution. Biomarkers of disease progression and therapeutic strategies based on these genes and mechanisms should be further investigated for this type of muscular dystrophy.This study was funded by Asociación Conquistando Escalones, French Agency for Research on AIDS and Viral Hepatitis (ANRS grant ECTZ107263), Instituto de Salud Carlos III (PI19CIII/00004), NIH grant R01AI143567, the Spanish Ministry of Science and Innovation (PID2019-110275RB I00) and Fundación Isabel Gemio. It has been conducted within the Spanish AIDS Research Network (RIS) and Centro de Investigación Biomédica en Red de Enfermedades Infecciosas, funded by Instituto de Salud Carlos 640 III (Plan Estatal de I+D+I 2013-2016) and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (RD16CIII/0002/0001).S

Role of ACE2 genetic polymorphisms in susceptibility to SARS-CoV-2 among highly exposed but non infected healthcare workers

We aim to evaluate the role of single-nucleotide polymorphisms of the angiotensin-converting enzyme 2 in susceptibility to SARS-CoV-2 infection. We included 28 uninfected but highly exposed healthcare workers and 39 hospitalized patients with COVID-19. Thirty-five SNPs were rationally selected. Two variants were associated with increased risk of being susceptible to SARS-CoV-2: the minor A allele in the rs2106806 variant (OR 3.75 [95% CI 1.23-11.43]) and the minor T allele in the rs6629110 variant (OR 3.39 [95% CI 1.09-10.56]). Evaluating the role of genetic variants in susceptibility to SARS-CoV-2 infection could help identify more vulnerable individuals and suggest potential drug targets for COVID-19 patients.This work was supported by Instituto de Salud Carlos III: [grant number AC17/00019,COV20/00349,PI18/00154,PT17/0019]; Merck, Sharp & Dohme: [Ref IISP 59181].S

The mutation of Transportin 3 gene that causes limb girdle muscular dystrophy 1F induces protection against HIV-1 infection

The causative mutation responsible for limb girdle muscular dystrophy 1F (LGMD1F) is one heterozygous single nucleotide deletion in the stop codon of the nuclear import factor Transportin 3 gene (TNPO3). This mutation causes a carboxy-terminal extension of 15 amino acids, producing a protein of unknown function (TNPO3_mut) that is co-expressed with wild-type TNPO3 (TNPO3_wt). TNPO3 has been involved in the nuclear transport of serine/arginine-rich proteins such as splicing factors and also in HIV-1 infection through interaction with the viral integrase and capsid. We analyzed the effect of TNPO3_mut on HIV-1 infection using PBMCs from patients with LGMD1F infected ex vivo. HIV-1 infection was drastically impaired in these cells and viral integration was reduced 16-fold. No significant effects on viral reverse transcription and episomal 2-LTR circles were observed suggesting that the integration of HIV-1 genome was restricted. This is the second genetic defect described after CCR5Δ32 that shows strong resistance against HIV-1 infection.This work was supported by crowfunding site PRECIPITA from FECYT, the MERCKSALUD Foundation, the Spanish Ministry of Science (FIS PI12/00969; PI16CIII/00034; SAF2016-78480-R); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (FEDER); CIBERer-ISCIII (FIS PI16/00316) co-financed by the European Regional Development Founds (FEDER), IIS La Fe (2016-0388; 2018-0200), and Fundación Isabel Gemio (http://www.fundacionisabelgemio.com). The work of Dra. Sara Rodríguez-Mora is supported by the Asociación Conquistando Escalones, funded by Spanish LGMD1F patients and Sara Borrell grant from Instituto de Salud Carlos III. The work of Dra. María Rosa López-Huertas is financed by ISCIII-Subdirección General de Evaluación and European Funding for Regional Development (FEDER) and by Spanish Ministry of Economy and Competitiveness (PIE13/00040). The work of Elena Mateos is supported by the Spanish Ministry of Economy and Competitiveness SAF2016-78480-R. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Long-Term Changes of Inflammatory Biomarkers in Individuals on Suppressive Three-Drug or Two-Drug Antiretroviral Regimens

Background: Because inflammation is associated with mortality and has been linked to HIV transcription in lymphoid tissues during ART, it is necessary to address the long-term effects of switching 3-drug (3DR) to 2-drug regimens (2DR) on inflammation. Methods: Nested study in the Spanish AIDS Research Network. We selected PWH ART-naive initiating 3DR who achieved viral suppression in the first 48 weeks and either remained on 3DR or switched to 2DR (3TC+bPI; 3TC+DTG; DTG+RPV). We assessed the trajectories on inflammatory markers during ART using multivariate piecewise mixed models. Results: We analyzed 619 plasma samples from 148 patients (3DR, N=90; 2DR, N=58), the median follow-up was 4.6 (IQR 3.2-6.2) years. There were no significant differences in baseline characteristics between groups. After adjusting for potential confounders, patients with 3DR experienced a slow decline of IL6, hs-CRP, sCD14, sCD163, and D-dimer over time. In contrast, compared to 3DR, switching to 2DR was associated with increases in IL-6 (p=0.001), hs-CRP (p=0.003), and D-dimer (p=0.001) after year 3 from virologic suppression. 2DR was associated with a higher risk of hs-CRP quartile increase (aOR 3.3, 95%CI 1.1-10) and D-dimer quartile increase (aOR 3.7, 95%CI 1.1-13). The adjusted biomarker trajectories did not reveal a distinct pattern according to the type of 2DR used (bPI vs DTG). Conclusions: In this study in virally suppressed individuals, maintaining 3DR was associated with a more favorable long-term inflammatory profile than switching to 2DR. The potential clinical implications of these findings on the development of non-AIDS events deserve further investigation.The HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by Instituto de Salud Carlos III, Spanish Health Ministry (Grant n° proyectos RD06/0006/0035, RD12/0017/0037 and RD16/0025/0019) as part of the Plan Nacional R + D + I and co-financed by the European Development Regional Fund ‘‘A way to achieve Europe’’ (ERDF). The RIS Cohort (CoRIS) is funded by the Instituto de Salud Carlos III through the Red Temática de Investigación Cooperativa en SIDA (RIS C03/173, RD12/0017/0018 and RD16/0002/0006) as part of the Plan Nacional R+D+I and cofinanced by ISCIII-Subdirección General de Evaluacion and Fondo Europeo de Desarrollo Regional (FEDER)”. This work was supported by the Instituto de Salud Carlos III projects AC17/00019, PI18/00154, ICI20/00058, CIBER de Enfermedades Infecciosas, and Gilead Sciences (Investigator Sponsored Research ISR-17-10192). The funders had no role in the study design, data analysis, or in the interpretation of the results.S

Association between HLA-C alleles and COVID-19 severity in a pilot study with a Spanish Mediterranean Caucasian cohort

The clinical presentations of COVID-19 may range from an asymptomatic or mild infection to a critical or fatal disease. Several host factors such as elderly age, male gender, and previous comorbidities seem to be involved in the most severe outcomes, but also an impaired immune response that causes a hyperinflammatory state but is unable to clear the infection. In order to get further understanding about this impaired immune response, we aimed to determine the association of specific HLA alleles with different clinical presentations of COVID-19. Therefore, we analyzed HLA Class I and II, as well as KIR gene sequences, in 72 individuals with Spanish Mediterranean Caucasian ethnicity who presented mild, severe, or critical COVID-19, according to their clinical characteristics and management. This cohort was recruited in Madrid (Spain) during the first and second pandemic waves between April and October 2020. There were no significant differences in HLA-A or HLA-B alleles among groups. However, despite the small sample size, we found that HLA-C alleles from group C1 HLA-C*08:02, -C*12:03, or -C*16:01 were more frequently associated in individuals with mild COVID-19 (43.8%) than in individuals with severe (8.3%; p = 0.0030; pc = 0.033) and critical (16.1%; p = 0.0014; pc = 0.0154) disease. C1 alleles are supposed to be highly efficient to present peptides to T cells, and HLA-C*12:03 may present a high number of verified epitopes from abundant SARS-CoV-2 proteins M, N, and S, thereby being allegedly able to trigger an efficient antiviral response. On the contrary, C2 alleles are usually poorly expressed on the cell surface due to low association with β2-microglobulin (β2M) and peptides, which may impede the adequate formation of stable HLA-C/β2M/peptide heterotrimers. Consequently, this pilot study described significant differences in the presence of specific HLA-C1 alleles in individuals with different clinical presentations of COVID-19, thereby suggesting that HLA haplotyping could be valuable to get further understanding in the underlying mechanisms of the impaired immune response during critical COVID-19.This work was supported by the Coordinated Research Activities at the Centro Nacional de Microbiologı´a (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); donation provided by Chiesi España, S.A.U. (Barcelona, Spain); the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00); and grant MPY509/19 provided by Instituto de Salud Carlos III. The work of MRLH and SRM is financed by NIH grant R01AI143567. The work of LV is supported by a pre-doctoral contract from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of MT is supported by Instituto de Salud Carlos III (COV20_00679). AJMG is the recipient of a post-doctoral contract“Miguel Servet” supported by the Instituto de Salud Carlos III.S

Cytotoxic cell populations developed during treatment with tyrosine kinase inhibitors protect autologous CD4+ T cells from HIV-1 infection

Factor de impacto: 5,858 Q1Tyrosine kinase inhibitors (TKIs) are successfully used in clinic to treat chronic myeloid leukemia (CML). Our group previously described that CD4+ T cells from patients with CML on treatment with TKIs such as dasatinib were resistant to HIV-1 infection ex vivo. The main mechanism for this antiviral activity was primarily based on the inhibition of SAMHD1 phosphorylation, which preserves the activity against HIV-1 of this innate immune factor. Approximately 50% CML patients who achieved a deep molecular response (DMR) may safely withdraw TKI treatment without molecular recurrence. Therefore, it has been speculated that TKIs may induce a potent antileukemic response that is maintained in most patients even one year after treatment interruption (TI). Subsequent to in vitro T-cell activation, we observed that SAMHD1 was phosphorylated in CD4+ T cells from CML patients who withdrew TKI treatment more than one year earlier, which indicated that these cells were now susceptible to HIV-1 infection. Importantly, these patients were seronegative for HIV-1 and seropositive for cytomegalovirus (CMV), but without CMV viremia. Although activated CD4+ T cells from CML patients on TI were apparently permissive to HIV-1 infection ex vivo, the frequency of proviral integration was reduced more than 12-fold on average when these cells were infected ex vivo in comparison with cells isolated from untreated, healthy donors. This reduced susceptibility to infection could be related to an enhanced NK-dependent cytotoxic activity, which was increased 8-fold on average when CD4+ T cells were infected ex vivo with HIV-1 in the presence of autologous NK cells. Enhanced cytotoxic activity was also observed in CD8 + T cells from these patients, which showed 8-fold increased expression of TCRγδ and more than 18-fold increased production of IFNγ upon activation with CMV peptides. In conclusion, treatment with TKIs induced a potent antileukemic response that may also have antiviral effects against HIV-1 and CMV, suggesting that transient use of TKIs in HIV-infected patients could develop a sustained antiviral response that would potentially interfere with HIV-1 reservoir dynamics.This work was supported by NIH grant R01AI143567; the Spanish Ministry of Economy and Competitiveness (SAF2016-78480-R); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of María Rosa López-Huertas and Sara Rodríguez-Mora is financed by NIH grant R01AI143567. The work of Lorena Vigón is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of Elena Mateos is supported by the Spanish Ministry of Economy and Competitiveness SAF2016-78480-R.S

Impaired Cytotoxic Response in PBMCs From Patients With COVID-19 Admitted to the ICU: Biomarkers to Predict Disease Severity

Infection by novel coronavirus SARS-CoV-2 causes different presentations of COVID-19 and some patients may progress to a critical, fatal form of the disease that requires their admission to ICU and invasive mechanical ventilation. In order to predict in advance which patients could be more susceptible to develop a critical form of COVID-19, it is essential to define the most adequate biomarkers. In this study, we analyzed several parameters related to the cellular immune response in blood samples from 109 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centers in Madrid, Spain, during the first pandemic peak between April and June 2020. Hospitalized patients with the most severe forms of COVID-19 showed a potent inflammatory response that was not translated into an efficient immune response. Despite the high levels of effector cytotoxic cell populations such as NK, NKT and CD8+ T cells, they displayed immune exhaustion markers and poor cytotoxic functionality against target cells infected with pseudotyped SARS-CoV-2 or cells lacking MHC class I molecules. Moreover, patients with critical COVID-19 showed low levels of the highly cytotoxic TCRγδ+ CD8+ T cell subpopulation. Conversely, CD4 count was greatly reduced in association to high levels of Tregs, low plasma IL-2 and impaired Th1 differentiation. The relative importance of these immunological parameters to predict COVID-19 severity was analyzed by Random Forest algorithm and we concluded that the most important features were related to an efficient cytotoxic response. Therefore, efforts to fight against SARS-CoV-2 infection should be focused not only to decrease the disproportionate inflammatory response, but also to elicit an efficient cytotoxic response against the infected cells and to reduce viral replication.This work was supported by the Coordinated Research Activities at the Centro Nacional de Microbiología (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr. Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain); the Spanish Ministry of Economy and Competitiveness (PID2019-110275RB-I00); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acciόn Estratégica en Salud, Plan Nacional de Investigaciόn Científica, Desarrollo e Innovaciόn Tecnolόgica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of ML-H and SR-M is financed by NIH grant R01AI143567. The work of LH is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER).S

Impaired Antibody-Dependent Cellular Cytotoxicity in a Spanish Cohort of Patients With COVID-19 Admitted to the ICU

SARS-CoV-2 infection causes COVID-19, ranging from mild to critical disease in symptomatic subjects. It is essential to better understand the immunologic responses occurring in patients with the most severe outcomes. In this study, parameters related to the humoral immune response elicited against SARS-CoV-2 were analysed in 61 patients with different presentations of COVID-19 who were recruited in Hospitals and Primary Healthcare Centres in Madrid, Spain, during the first pandemic peak between April and June 2020. Subjects were allocated as mild patients without hospitalization, severe patients hospitalized or critical patients requiring ICU assistance. Critical patients showed significantly enhanced levels of B cells with memory and plasmablast phenotypes, as well as higher levels of antibodies against SARS-CoV-2 with neutralization ability, which were particularly increased in male gender. Despite all this, antibody-dependent cell-mediated cytotoxicity was defective in these individuals. Besides, patients with critical COVID-19 also showed increased IgG levels against herpesvirus such as CMV, EBV, HSV-1 and VZV, as well as detectable CMV and EBV viremia in plasma. Altogether, these results suggest an enhanced but ineffectual immune response in patients with critical COVID-19 that allowed latent herpesvirus reactivation. These findings should be considered during the clinical management of these patients due to the potential contribution to the most severe disease during SARS-CoV-2 infection.This work was supported by the Coordinated Research Activities at the Centro Nacional de Microbiología (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation) that is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM) and a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. This work was also supported by the Spanish Ministry of Economy and Competitiveness (PID2019 110275RB-I00); the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF); Miguel Servet - AESI, MPY 341/21. The work of ML-H and SR is financed by NIH grant R01AI143567. The work of MT is supported by Instituto de Salud Carlos III (COV20_00679). The work of LV is supported by a predoctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER).S