Envejecimiento prematuro en pacientes VIH+: evaluación in vitro e in vivo del impacto del tratamiento con análogos de nucleós(t)idos en la longitud de los telómeros

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-01-2019Gracias al indiscutible éxito del tratamiento antirretroviral (TAR) la esperanza de vida del paciente VIH ha aumentado significativamente hasta aproximarse a la de la persona no infectada. Sin embargo, el TAR no consigue restaurar por completo la salud del paciente por lo que la población VIH+ presenta múltiples comorbilidades asociadas al envejecimiento a edades menos avanzadas que en la población no VIH. Dado que en población general existe una asociación entre las enfermedades asociadas al envejecimiento y el acortamiento telomérico, y además existe una similitud estructural y funcional entre la transcriptasa inversa del VIH y la telomerasa celular, el trabajo realizado en esta tesis doctoral se ha centrado en el estudio del impacto de los inhibidores de la transcriptasa inversa análogos de nucleós(t)idos (N(t)RTIs) en la actividad telomerasa in vitro y su impacto en la longitud telomérica (LT) in vivo tanto en pacientes con prolongada supresión virológica como pacientes naïve que comienzan tratamiento antirretroviral (TAR) de primera línea. En el estudio in vitro hemos confirmado que concentraciones terapéuticas de los N(t)RTIs tenofovir (TFV), principio activo del fármaco antirretroviral tenofovir difumarato (TDF), y abacavir (ABC) pero no de emtricitabina (FTC) producen una disminución de la actividad telomerasa dosis-dependiente, siendo TFV el inhibidor más potente. Esta inhibición no se asocia a cambios en los niveles de proteína ni de la expresión génica de los componentes de la telomerasa o del complejo shelterina. En los estudios in vivo, realizados tanto en la cohorte de pacientes con supresión virológica prolongada como en los pacientes naïve que comienzan TAR de primera línea, se observó de forma global una ganancia de la LT en sangre tras dos años de seguimiento. Al analizar la LT en función del régimen del TAR, en el caso de los pacientes VIH+ con prolongada supresión viral la LT en sangre aumentó menos en el grupo de pacientes expuestos a TDF o ABC que en el grupo tratado con la pauta ahorradora de N(t)RTIs. Por el contrario, en los pacientes naïve la LT en sangre aumentó más en el grupo de pacientes tratados con TDF/TFC que en el grupo tratado con la pauta ahorradora de N(t)RTIs. Estas diferencias sugieren que los N(t)RTIs producen una mejor recuperación inicial de la inmunosenescencia asociada a la infección por el VIH; sin embargo interfieren con la recuperación a largo plazo, posiblemente debido a su capacidad para inhibir la telomerasa.Due to the success of antiretroviral treatment (ART), the life expectancy of the HIV patient has increased significantly, being close to the uninfected person. However, ART does not fully restore the health of patient, so the HIV population has multiple comorbidities associated with aging at less advanced ages than the non-HIV population. Given that in the general population there is an association between diseases associated with aging and telomeric shortening, and there is also a structural and functional similarity between HIV reverse transcriptase and cellular telomerase, the work carried out in this doctoral thesis has focused on the study of the impact of nucleoside and nucleotide reverse transcriptase inhibitors (N(t)RTIs) on telomerase activity in vitro and its impact on telomere length (TL) in vivo in patients with prolonged virological suppression as well as naïve patients who start first-line antiretroviral (ART) treatment. In the in vitro study we have confirmed that therapeutic concentrations of the N(t)RTIs such as tenofovir (TFV), the active substance of the antiretroviral drug tenofovir difumarate (TDF), and abacavir (ABC) but not of emtricitabine (FTC) produce a decrease in dose-dependent telomerase activity, TFV being the most potent inhibitor. This inhibition is not associated with changes in protein levels or gene expression of the components of telomerase or shelterin complex. In in vivo studies, carried out both in the cohort of patients with prolonged virological suppression and in naïve patients who started first-line ART, a gain in TL in blood was observed overall after two years of follow-up. When TL was analyzed according to the ART regimen, in the case of HIV patients with prolonged viral suppression, blood TL increased less in the group of patients exposed to TDF or ABC than in the group treated with the N(t)RTIs-sparing regimen. Conversely, in naïve patients the TL in blood increased more in the group of patients treated with TDF/TFC than in the group treated with the N(t)RTIs-sparing regimen. These differences suggest that N(t)RTIs produce a better initial recovery of immunosenescence associated with HIV infection; however, they interfere with long-term recovery, possibly due to their ability to inhibit telomerase

: PLoS One

International audienceIn 31 participants who started first-line antiretroviral therapy in the NEAT 001/ANRS 143 clinical trial, we found after 96 weeks a statistically significant increase in blood telomere length (TL) of 0.04 (T/S Ratio) (p = 0.03). This increase was positively correlated with both the change in the percentage of CD4+ T-cells and with the decrease of CD38+ molecules on Central Memory CD8+ and negatively correlated with the change in the percentage of CD4+ Effector Memory cells. Increase in TL could be an expression of immune reconstitution and the associated decrease in immune activation. We acknowledge for the low statistical power due to the small sample size and the potential for false positive results due to multiple testing. Hence, further studies are needed to confirm these observations

Brief report: Differential effects of tenofovir, abacavir, emtricitabine, and darunavir on telomerase activity in vitro

In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in peripheral blood mononuclear cells of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5 and 1 M was 29% [Interquartile range (IQR) 29%-34%, P = 0.042] and 28% (IQR 28%-41%, P = 0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, P = 0.043) at 3 M and 14% (IQR 10%-29%, P = 0.043) at 10 M. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels, or mRNA levels of telomerase/shelterin genes.Peer Reviewe

Dolutegravir plus lamivudine for maintenance of HIV viral suppression in adults with and without historical resistance to lamivudine: 48-week results of a non-randomized, pilot clinical trial (ART-PRO)

Background We investigated the efficacy of a switch to dolutegravir plus lamivudine in aviremic individuals without evidence of persistent lamivudine resistance-associated mutations in baseline proviral DNA population sequencing. Methods Open-label, single-arm, 48-week pilot trial. HIV-1 infected adults, naïve to integrase inhibitors, with CD4+ above 350 cell/μL and fewer than 50 HIV-1 RNA copies per mL the year prior to study entry switched to dolutegravir plus lamivudine. Participants were excluded if baseline proviral DNA population genotyping detected lamivudine resistance-associated mutations. To detect resistance minority variants, proviral DNA next-generation sequencing was retrospectively performed from baseline samples. Primary efficacy endpoint was proportion of participants with fewer than 50 HIV-1 RNA copies per mL at week 48. Safety and tolerability outcomes were incidence of adverse events and treatment discontinuations. ART-PRO is registered with ClinicalTrials.gov, NCT03539224. Findings 41 participants switched to dolutegravir plus lamivudine, 21 with lamivudine resistance mutations in historical plasma genotypes. Baseline next-generation sequencing detected lamivudine resistance mutations (M184V/I and/or K65R/E/N) over a 5% threshold in 15/21 (71·4%) and 3/20 (15%) of participants with and without history of lamivudine resistance, respectively. At week 48, 92·7% of participants (38/41) had fewer than 50 HIV-1 RNA copies per mL. There were no cases of virologic failure. Three participants with historical lamivudine resistance were prematurely discontinued from the study (2 protocol violations, one adverse event). Ten participants (4 in the group with historical lamivudine resistance) had a transient viral rebound, all resuppressed on dolutegravir plus lamivudine. There were 28 drug-related adverse events, only one leading to discontinuation. Interpretation In this pilot trial, dolutegravir plus lamivudine was effective in maintaining virologic control despite past historical lamivudine resistance and presence of archived lamivudine resistance-associated mutations detected by next generation sequencing. Further studies are needed to confirm our results.Instituto de Salud Carlos III PI16/00837-PI16/006788.143 JCR (2020) Q1, 17/140 Medicine, Research & Experimental2.596 SJR (2020) Q1, 23/254 Biochemistry, Genetics and Molecular Biology (miscellaneous)No data IDR 2020UE

Dolutegravir plus lamivudine for maintenance of HIV viral suppression in adults with and without historical resistance to lamivudine: 48-week results of a non-randomized, pilot clinical trial (ART-PRO).

We investigated the efficacy of a switch to dolutegravir plus lamivudine in aviremic individuals without evidence of persistent lamivudine resistance-associated mutations in baseline proviral DNA population sequencing. Open-label, single-arm, 48-week pilot trial. HIV-1 infected adults, naïve to integrase inhibitors, with CD4+ above 350 cell/μL and fewer than 50 HIV-1 RNA copies per mL the year prior to study entry switched to dolutegravir plus lamivudine. Participants were excluded if baseline proviral DNA population genotyping detected lamivudine resistance-associated mutations. To detect resistance minority variants, proviral DNA next-generation sequencing was retrospectively performed from baseline samples. Primary efficacy endpoint was proportion of participants with fewer than 50 HIV-1 RNA copies per mL at week 48. Safety and tolerability outcomes were incidence of adverse events and treatment discontinuations. ART-PRO is registered with ClinicalTrials.gov, NCT03539224. 41 participants switched to dolutegravir plus lamivudine, 21 with lamivudine resistance mutations in historical plasma genotypes. Baseline next-generation sequencing detected lamivudine resistance mutations (M184V/I and/or K65R/E/N) over a 5% threshold in 15/21 (71·4%) and 3/20 (15%) of participants with and without history of lamivudine resistance, respectively. At week 48, 92·7% of participants (38/41) had fewer than 50 HIV-1 RNA copies per mL. There were no cases of virologic failure. Three participants with historical lamivudine resistance were prematurely discontinued from the study (2 protocol violations, one adverse event). Ten participants (4 in the group with historical lamivudine resistance) had a transient viral rebound, all resuppressed on dolutegravir plus lamivudine. There were 28 drug-related adverse events, only one leading to discontinuation. In this pilot trial, dolutegravir plus lamivudine was effective in maintaining virologic control despite past historical lamivudine resistance and presence of archived lamivudine resistance-associated mutations detected by next generation sequencing. Further studies are needed to confirm our results. Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III PI16/00837-PI16/00678.Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III PI16/00837-PI16/00678.S

Correlation between blood telomere length and CD4+ CD8+ T-cell subsets changes 96 weeks after initiation of antiretroviral therapy in HIV-1-positive individuals

In 31 participants who started first-line antiretroviral therapy in the NEAT 001/ANRS 143 clinical trial, we found after 96 weeks a statistically significant increase in blood telomere length (TL) of 0.04 (T/S Ratio) (p = 0.03). This increase was positively correlated with both the change in the percentage of CD4+ T-cells and with the decrease of CD38+ molecules on Central Memory CD8+ and negatively correlated with the change in the percentage of CD4+ Effector Memory cells. Increase in TL could be an expression of immune reconstitution and the associated decrease in immune activation. We acknowledge for the low statistical power due to the small sample size and the potential for false positive results due to multiple testing. Hence, further studies are needed to confirm these observations

Impact of antiretroviral treatment containing tenofovir difumarate on the telomere length of aviremic HIV-infected patients

[Objective]: To evaluate the in vivo relevance of the inhibitory effect of tenofovir on telomerase activity observed in vitro.[Design]: Cross-sectional study of HIV-infected patients with suppressed virological replication (HIV RNA <50 copies/mL for more than 1 year).[Methods]: Telomere length in whole blood was measured by quantitative real-time polymerase chain reaction. We performed a multivariate analysis to elucidate variables associated with telomere length and also evaluated the association between telomere length and use of tenofovir difumarate (TDF) adjusted by significant confounders.[Results]: 200 patients included, 72% men, median age 49 (IQR 45–54.5), 103 with exposure to a TDF containing antiretroviral treatment (ART) regimen (69.9% for more than 5 years) and 97 never exposed to a TDF containing ART regimen. In the multivariate analysis, significant predictors of shorter telomere length were older age (P = 0.008), parental age at birth (P = 0.038), white race (P = 0.048), and longer time of known HIV infection (10–20 and ≥20 years compared with <10 years, P = 0.003 and P = 0.056, respectively). There was no association between TDF exposure and telomere length after adjusting for possible confounding factors (age, parental age at birth, race, and time of HIV infection). Total time receiving ART and duration of treatment with nucleoside reverse transcriptase inhibitors were associated with shorter telomere length, but these associations were explained by time of known HIV infection.[Conclusions]: Our data do not suggest that telomerase activity inhibition caused by TDF in vitro leads to telomere shortening in peripheral blood of HIV-infected patients.This work was supported by grant PI13/01467 and PI14/01495 from Fondo de Investigaciones Sanitarias (supported by FEDER funds). Instituto de Salud Carlos III. Natalia Stella is supported by a predoctoral fellowship from Fondo de Investigaciones Sanitarias. Rocio Montejano is supported by a Río Hortega fellowship from Fondo de Investigaciones Sanitarias.Peer reviewe