Epigenetic age acceleration changes 2 years after antiretroviral therapy initiation in adults with HIV: a substudy of the NEAT001/ANRS143 randomised trial

doi:10.1016/S2352-3018(21)00006-0

The Lancet HIV

Volume 8, Issue 4, April 2021, Pages e197-e205

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https://doi.org/10.1016/S2352-3018(21)00006-0 Get rights and content

Summary

Background

DNA methylation-based estimators of biological age are reliable biomarkers of the ageing process. We aimed to investigate a range of epigenetic ageing biomarkers in a substudy of the NEAT001/ANRS143 clinical trial, which compared ritonavir-boosted darunavir with either raltegravir or tenofovir disoproxil fumarate and emtricitabine in antiretroviral therapy (ART)-naive adults.

Methods

We analysed frozen whole blood samples from 168 ART-naive participants with HIV from the NEAT001/ANRS143 trial, before ART initiation and after 2 years of ART (84 participants on ritonavir-boosted darunavir with raltegravir and 84 participants on ritonavir-boosted darunavir with tenofovir disoproxil fumarate and emtricitabine). We also included 44 participants without HIV with a similar age and sex distribution. We analysed DNA methylation. Epigenetic age estimators (Horvath's clock, Hannum's clock, GrimAge, and PhenoAge) and estimated leucocyte compositions were generated using Horvath's New Online Methylation Age Calculator and Houseman's method. We calculated epigenetic age acceleration measures for each estimator of epigenetic age. The NEAT001/ANRS143 trial is registered with ClinicalTrials.gov, NCT01066962.

Findings

Compared with the HIV-uninfected group, ART-naive participants with HIV showed higher epigenetic age acceleration (EAA) according to all EAA estimators (mean 2·5 years, 95% CI 1·89–3·22 for Horvath-EAA; 1·4 years, 0·74–1·99 for Hannum-EAA; 2·8 years, 1·97–3·68 for GrimAge-EAA; and 7·3 years, 6·40–8·13 for PhenoAge-EAA), with all differences being statistically significant except for Hannum-EAA (Horvath-EAA p=0·0008; Hannum-EAA p=0·059; GrimAge-EAA p=0·0021; and PhenoAge-EAA p<0·0001). Epigenetic ageing was more pronounced in participants who had CD4 counts less than 200 cells per μL (significant for PhenoAge and Hannum's clock, p=0·0015 and p=0·034, respectively) or viral loads over 100 000 copies per mL at baseline (significant for PhenoAge, p=0·017). After 2 years of ART, epigenetic age acceleration was reduced, although PhenoAge and GrimAge remained significantly higher in participants with HIV compared with participants without HIV (mean difference 3·69 years, 95% CI 1·77–5·61; p=0·0002 and 2·2 years, 0·47–3·99; p=0·013, respectively). There were no significant differences in the ART effect on epigenetic ageing between treatment regimens. At baseline, participants with HIV showed dysregulation of DNA methylation-based estimated leucocyte subsets towards more differentiated T-cell phenotypes and proinflammatory leucocytes, which was also partly restored with ART.

Interpretation

ART initiation partly reversed epigenetic ageing associated with untreated HIV infection. Further studies are needed to understand the long-term dynamics and clinical relevance of epigenetic ageing biomarkers in people with HIV.

Funding

NEAT-ID Foundation.

Introduction

Antiretroviral therapy (ART) has transformed HIV infection into a chronic and manageable disease in which life expectancy among people living with HIV approaches that of the general population.¹ However, this prolonged survival in people living with HIV has been linked to an increased burden of age-related comorbidities, such as cardiovascular disease, cancer, cognitive impairment, osteoporosis, and frailty.2, 3 Although the cause of this accelerated or accentuated ageing is not completely understood, it might be related to residual immunosenescence and inflammation that persists despite successful ART.4, 5

Until roughly the past 5 years, the study of ageing and its consequences lacked precise and reproducible biomarkers. Although blood telomere length is negatively associated with chronological age, its ability to predict life expectancy is restricted.⁶ Among the most reliable biomarkers of age are so-called epigenetic clocks, mathematical algorithms that predict epigenetic age as a surrogate of biological age based on the DNA methylation levels of different sets of CpG dinucleotide sites in the genome that are known to change with ageing.⁷ The first to be developed were Horvath's multi-tissue epigenetic clock, based on 353 CpG sites,⁸ and Hannum's epigenetic clock for blood samples, based on 71 CpG sites.⁹ These epigenetic clocks are attractive biomarkers of ageing because they have a strong correlation with chronological age and because epigenetic age acceleration (EAA), defined as an epigenetic age greater than that predicted based on chronological age, predicts the occurrence of age-related comorbidities and mortality.7, 10 In the past two years, two new DNA methylation-based estimators of ageing have been developed: PhenoAge and GrimAge. PhenoAge predicts a surrogate measure of phenotypic age based on 513 CpG sites, and is considered an accurate predictor of mortality, healthspan (ie, the period of time in which a person is in good health), cardiovascular disease, and other morbidities.¹¹ The GrimAge estimator, which is calculated based on chronological age, sex, and DNA methylation-based surrogates for seven plasma proteins and smoking pack-years, also predicts time to death and comorbidities.¹² There are almost no data concerning the evolution of these biomarkers after successful treatment of diseases that shorten the lifespan. An example of such a disease is HIV, which has a median survival from age 25 of 19·9 years when untreated, compared with 51·1 years for people without HIV.¹³

Research in context

Evidence before this study

Data on the effect of HIV and antiretroviral therapy (ART) on epigenetic biomarkers of ageing are scarce. We searched PubMed for reports published in English, with no restrictions on publication date, using combinations of the following keywords: “HIV infection”, “antiretroviral therapy”, “premature aging”, “epigenetic clocks”, “epigenetic aging”, and “epigenetic age acceleration”. We also searched for relevant publications from international HIV congresses (2017–20) using the same search criteria. Our search yielded six publications. Five of these reports found an association between HIV infection and epigenetic age acceleration (EAA), both in untreated and treated HIV infection. Nevertheless, none of these studies evaluated the effect of ART on epigenetic ageing in treatment-naive adults with HIV in the context of a clinical trial. We found only one longitudinal study that reported a positive effect of ART initiation on epigenetic ageing in people with HIV. However, this study included a small number of participants (n=19) and was not fully powered to establish how epigenetic ageing dynamics change immediately after introducing ART.

Added value of this study

To our knowledge, this is the first study to assess changes in biomarkers of epigenetic ageing after ART initiation in a population of participants with HIV enrolled in a clinical trial (NEAT001/ANRS143). Our results support evidence that untreated HIV infection is associated with EAA, which is more pronounced in participants with severe immunodeficiency. Our study also suggests that ART partly reverses epigenetic ageing only 2 years after initiation. We also compared, for the first time to our knowledge, the effect of different ART regimens on epigenetic ageing dynamics. We found no significant difference in epigenetic ageing reversal between participants receiving darunavir and ritonavir plus raltegravir or darunavir and ritonavir plus tenofovir disoproxil fumarate and emtricitabine regimens.

Implications of all the available evidence

To our knowledge, our study is one of the first examples of how biomarkers of epigenetic ageing can capture the initial beneficial effect of a therapeutic intervention that significantly prolongs lifespan. The partial reversal of HIV-induced EAA supports an additional beneficial effect of ART. EAA predicts a higher risk of emergence of comorbidities and mortality in the general population, but its clinical relevance in people living with HIV remains to be appropriately defined. More evidence is needed to elucidate whether biomarkers of epigenetic ageing could help to identify people with chronic HIV who are more likely to suffer premature age-related comorbidities.

Previous studies have reported that HIV is associated with accelerated epigenetic ageing in adults and perinatally HIV-infected children on ART.14, 15, 16 Furthermore, in a cohort of 31 injection drug users, this accelerated ageing started soon after HIV seroconversion.¹⁷ However, there is a paucity of data concerning the effect of ART and specific antiretroviral regimens on the evolution of epigenetic ageing. We aimed to investigate, for the first time to our knowledge, a range of epigenetic ageing biomarkers in a substudy of the NEAT001/ANRS143 clinical trial, which compared ritonavir-boosted darunavir combined with either raltegravir or tenofovir disoproxil fumarate and emtricitabine in ART-naive adults.¹⁸ We hypothesised that ART would have a beneficial effect on epigenetic ageing.

Section snippets

Study design and participants

We included participants from the NEAT001/ANRS143 clinical trial. Briefly, NEAT001/ANRS143 was a randomised, open-label, non-inferiority trial at 78 clinical sites in 15 European countries (appendix pp 6–7) between August, 2010, and October, 2013. The trial showed non-inferiority over 96 weeks of ritonavir-boosted darunavir combined with raltegravir versus ritonavir-boosted darunavir combined with tenofovir disoproxil fumarate and emtricitabine in ART-naive adults aged ≥18 years with HIV.¹⁸ For

Results

In participants with HIV, baseline characteristics were similar between treatment groups, with the exception of the nadir CD4 count (table). At week 96, the immunological and virological responses did not differ between the treatment groups. 159 (95%) of 168 participants had virological suppression and the median CD4 to CD8 ratio increased after ART initiation (table). Participants with HIV and HIV-uninfected individuals were well balanced for age (median 38·1 years, IQR 30·5–46·5 vs 39 years,

Discussion

In this study, we found that ART-naive adults with HIV had accelerated epigenetic ageing, which was more pronounced in those with CD4 counts less than 200 cells per μL or viral loads over 100 000 copies per mL. We observed a partial reversal of epigenetic ageing after only 2 years of ART, with no difference between participants on ritonavir-boosted darunavir with raltegravir or ritonavir-boosted darunavir with tenofovir disoproxil fumarate and emtricitabine. However, it is possible that other

Data sharing

De-identified individual participant data that underlie the results reported in this Article will be made available upon request to the corresponding author.

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Epigenetic ageing accelerates before antiretroviral therapy and decelerates after viral suppression in people with HIV in Switzerland: a longitudinal study over 17 years
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Accelerated epigenetic ageing can occur in untreated HIV infection and is partially reversible with effective antiretroviral therapy (ART). We aimed to make a long-term comparison of epigenetic ageing dynamics in people with HIV during untreated HIV infection and during suppressive ART.
In this longitudinal study, conducted over 17 years in HIV outpatient clinics in Switzerland, we applied 5 established epigenetic age estimators (epigenetic clocks) in peripheral blood mononuclear cells (PBMCs) in Swiss HIV Cohort Study participants before or during suppressive ART. All participants had a longitudinal set of PBMC samples available at four timepoints (T1–T4). T1 and T2 had to be 3 years or longer apart, as did T3 and T4. We assessed epigenetic age acceleration (EAA) and a novel rate of epigenetic ageing.
Between March 13, 1990, and Jan 18, 2018, we recruited 81 people with HIV from the Swiss HIV Cohort Study. We excluded one participant because a sample did not meet quality checks (transmission error). 52 (65%) of 80 patients were men, 76 (95%) were white, and the median patient age was 43 (IQR 37·5–47) years. Per year of untreated HIV infection (median observation 8·08 years, IQR 4·83–11·09), mean EAA was 0·47 years (95% CI 0·37 to 0·57) for Horvath's clock, 0·43 years (0·3 to 0·57) for Hannum's clock, 0·36 years (0·27 to 0·44) for SkinBlood clock, and 0·69 years (0·51 to 0·86) for PhenoAge. Per year of suppressive ART (median observation 9·8 years, IQR 7·2–11), mean EAA was –0·35 years (95% CI –0·44 to –0·27) for Horvath's clock, –0·39 years (–0·50 to –0·27) for Hannum's clock, –0·26 years (–0·33 to –0·18) for SkinBlood clock, and –0·49 years (–0·64 to –0·35) for PhenoAge. Our findings indicate that people with HIV epigenetically aged by a mean of 1·47 years for Horvath's clock, 1·43 years for Hannum's clock, 1·36 years for SkinBlood clock, and 1·69 years for PhenoAge per year of untreated HIV infection; and 0·65 years for Horvath's clock, 0·61 years for Hannum's clock, 0·74 years for SkinBlood clock, and 0·51 years for PhenoAge, per year of suppressive ART. GrimAge showed some change in the mean EAA during untreated HIV infection (0·10 years, 0·02 to 0·19) and suppressive ART (–0·05 years, –0·12 to 0·02). We obtained very similar results using the rate of epigenetic ageing. Contribution of multiple HIV-related, antiretroviral, and immunological variables, and of a DNA methylation-associated polygenic risk score to EAA was small.
In a longitudinal study over more than 17 years, epigenetic ageing accelerated during untreated HIV infection and decelerated during suppressive ART, highlighting the importance of limiting the duration of untreated HIV infection.
Swiss HIV Cohort Study, Swiss National Science Foundation, and Gilead Sciences.
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Citation Excerpt :
Eliminating inflammation or the trigger of inflammation, e.g. with antiviral therapy, may slow, halt or even reverse biological aging. Recently, it was shown that individuals infected with HIV showed improvement in epigenetic age after 96 weeks of antiretroviral therapy.33 Gindin et al. showed that one-year antiviral treatment of chronic hepatitis B was associated with a modest reduction in age acceleration.30
Chronic hepatitis C virus (HCV) infection can be cured with direct-acting antivirals (DAAs). However, not all sequelae of chronic hepatitis C appear to be completely reversible after sustained virologic response (SVR). Recently, chronic viral infections have been shown to be associated with biological age acceleration defined by the epigenetic clock. The aim of this study was to investigate whether chronic HCV infection is associated with epigenetic changes and biological age acceleration and whether this is reversible after SVR.
We included 54 well-characterized individuals with chronic hepatitis C who achieved SVR after DAA therapy at three time points: DAA treatment initiation, end of treatment, and long-term follow-up (median 96 weeks after end of treatment). Genome-wide DNA methylation status was determined in peripheral blood mononuclear cells (PBMCs) and used to calculate epigenetic age acceleration (EAA) using Horvath’s clock.
Individuals with HCV had an overall significant EAA of 3.12 years at baseline compared with -2.61 years in the age- and sex-matched reference group (p <0.00003). HCV elimination resulted in a significant long-term increase in DNA methylation dominated by hypermethylated CpGs in all patient groups. Accordingly, EAA decreased to 1.37 years at long-term follow-up. The decrease in EAA was significant only between the end of treatment and follow-up (p = 0.01). Interestingly, eight individuals who developed hepatocellular carcinoma after SVR had the highest EAA and showed no evidence of reversal after SVR.
Our data contribute to the understanding of the biological impact of HCV elimination after DAA therapy and demonstrate that HCV elimination can lead to “reverse inflammaging”. In addition, our data support the potential use of biological age as a biomarker for HCV sequelae after SVR.
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View all citing articles on Scopus

^*: Contributed equally

^‡: Both contributed equally

^§: Members are listed in the appendix pp 4–8

^†: Denes Banhegyi died in October, 2019

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ArticlesEpigenetic age acceleration changes 2 years after antiretroviral therapy initiation in adults with HIV: a substudy of the NEAT001/ANRS143 randomised trial

Summary

Background

Methods

Findings

Interpretation

Funding

Introduction

Section snippets

Study design and participants

Results

Discussion

Data sharing

Semin Diagn Pathol

Mol Cell

Mol Cell

Lancet

Premature age-related comorbidities among HIV-infected persons compared with the general population

Clin Infect Dis

HIV infection, inflammation, immunosenescence, and aging

Annu Rev Med

Systemic inflammation and the increased risk of inflamm-aging and age-associated diseases in people living with HIV on long term suppressive antiretroviral therapy

Front Immunol

Is telomere length a biomarker of aging? A review

J Gerontol A Biol Sci Med Sci

DNA methylation-based biomarkers and the epigenetic clock theory of ageing

Nat Rev Genet

DNA methylation age of human tissues and cell types

Genome Biol

DNA methylation-based measures of biological age: meta-analysis predicting time to death

Aging (Albany NY)

An epigenetic biomarker of aging for lifespan and healthspan

Aging (Albany NY)

DNA methylation GrimAge strongly predicts lifespan and healthspan

Aging (Albany NY)

Survival of persons with and without HIV infection in Denmark, 1995–2005

Ann Intern Med

Articles
Epigenetic age acceleration changes 2 years after antiretroviral therapy initiation in adults with HIV: a substudy of the NEAT001/ANRS143 randomised trial