HIV-DNA and Risk of HIV-RNA Rebound After Sustained Suppression

Background and Rationale

Combination antiretroviral therapy (cART) has transformed Human Immunodeficiency Virus type 1 (HIV-1) infection into a manageable chronic disease, achieving plasma HIV-RNA suppression below detection limits (typically <50 copies/mL) in over 95% of adherent patients. However, cART does not eradicate the virus. HIV persists in long-lived latently infected CD4+ T-cell reservoirs and anatomical sanctuaries. This persistent viral reservoir represents the major obstacle to a sterilizing or functional HIV cure and remains a continuous source of low-level viral dynamics, chronic immune activation, and systemic inflammation.

While plasma HIV-RNA monitoring is the current gold standard for assessing ART efficacy, it provides no direct information regarding the magnitude, composition, or dynamics of the underlying proviral reservoir. Quantifying total HIV-DNA in peripheral blood mononuclear cells (PBMCs) or purified CD4+ T cells serves as a widely established surrogate marker of reservoir size. High baseline or longitudinal total HIV-DNA levels have been linked to higher risks of virological rebound, blunted CD4+ T-cell recovery, and persistent immune dysregulation.

However, total HIV-DNA measurements do not distinguish between replication-competent proviruses and defective proviral genomes (which constitute >90-95% of the total HIV proviral pool in individuals on long-term cART). To address this limitation, the Intact Proviral DNA Assay (IPDA) was developed as a high-throughput digital droplet PCR (ddPCR)-based method capable of selectively quantifying intact, potentially replication-competent proviruses by targeting highly conserved regions (env and packaging signal/psi) while discriminating defective proviruses containing large deletions or hypermutations.

Despite the biological relevance of intact proviral DNA, the exact clinical utility and predictive accuracy of both total HIV-DNA and intact proviral DNA as actionable clinical biomarkers remain to be fully consolidated in real-world clinical cohorts.

Specifically, there is a critical need to evaluate whether quantification of total HIV-DNA and intact proviral DNA during sustained plasma viral suppression can accurately stratify people living with HIV (PWH) according to their risk of subsequent viral rebound. Furthermore, understanding the interplay between proviral reservoir dynamics and chronic systemic inflammation (measured via soluble inflammatory biomarkers) is vital for improving long-term clinical management and health outcomes in PWH.

Study Objectives Primary Objective. To establish and standardize a multi-center biomarker platform across participating European clinical research sites and evaluate whether total HIV-DNA and intact proviral DNA (IPDA) levels measured during sustained plasma HIV-RNA suppression predict the risk of subsequent HIV-RNA virological rebound in PWH on modern cART.

Secondary Objectives:

• To compare the predictive performance and accuracy of total HIV-DNA vs. intact proviral DNA (IPDA) as biomarkers for virological rebound.
• To assess the correlation between total/intact HIV-DNA quantification and plasma levels of systemic inflammatory and immune activation markers (e.g., IL-6, sCD14, sCD163, IP-10, hs-CRP) during viral suppression.
• To evaluate the impact of clinical, demographic, and therapeutic variables (e.g., nadir CD4+ T-cell count, pre-ART HIV-RNA, cART regimen duration and anchor drug class, HIV-1 subtype) on proviral reservoir size and viral rebound risk.
• To characterize the frequency and structural nature of defective proviruses in PWH experiencing viral rebound compared to continuously suppressed controls.

Study Design and Methodology Overall Design. This is an international, multi-center, retrospective and prospective observational case-control study conducted as part of Work Package 3 (WP3-HIV) of the Horizon Europe / EU-funded VIROMARKERS project (Grant Agreement No. 101194735).

The study leverages well-characterized, prospective clinical cohorts of PWH in Europe, including:

Italian Cohort Naive Antiretrovirals (I.Co.Na); ASST Santi Paolo e Carlo / University of Milan Cohort (Milan, Italy); Karolinska Institutet Cohort (Stockholm, Sweden); University Hospital Düsseldorf / Bonn Cohort (Germany); University Hospital Cologne (UniKoeln) Cohort (Cologne, Germany) ;

Study Population and Group Allocation. The study will evaluate a minimum of 300 PWH partitioned into two groups in a 1:2 (Case:Control) ratio.

Cases (n = 100): PWH under sustained viral suppression on modern cART who subsequently experience virological rebound, defined as:

Two consecutive plasma HIV-RNA measurements >200 copies/mL, or a single plasma HIV-RNA measurement >1,000 copies/mL leading to a change in antiretroviral regimen.

Controls (n = 200): PWH continuously suppressed (HIV-RNA ≤50 copies/mL) over an equivalent follow-up period, matched 2:1 to cases based on:

age (±5 years), biological sex, specific clinical cohort, duration of prior virological suppression (±6 months).

Eligibility Criteria

Inclusion Criteria. Age ≥ 18 years at the time of study enrolment. Confirmed HIV-1 infection. Enrolment in a participating prospective clinical cohort with documented regular clinical and virological follow-up (minimum 2 HIV-RNA viral load measurements per year).

Sustained plasma HIV-RNA suppression (≤50 copies/mL) for at least 6 consecutive months on modern cART (regimen initiated after 2012).

Availability of stored biological samples (PBMCs, whole blood, and matching plasma) collected during virological suppression (at a baseline timepoint at least 3 months prior to virological rebound for cases).

At least 2 available plasma HIV-RNA measurements during post-baseline follow-up.

Written informed consent provided according to institutional and national regulatory guidelines.

Exclusion Criteria. Active, untreated Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV) coinfection (presence of HBsAg positive or HCV-RNA positive).

Active opportunistic infections, active malignancy, or systemic autoimmune disorders at the time of sample collection.

Concurrent administration of systemic immunomodulatory or immunosuppressive therapies (e.g., prolonged corticosteroid therapy, chemotherapy).

Laboratory Procedures and Biomarker Assays Sample Collection and Processing. Stored PBMC, whole blood, and plasma samples collected during documented viral suppression will be retrieved from biobanks at participating sites and processed under standardized protocol specifications.

Total HIV-DNA Quantification:

genomic DNA is extracted from whole blood or frozen PBMC aliquots using standardized automated extraction protocols. Total HIV-DNA is quantified using digital PCR (dPCR) or quantitative real-time PCR (qPCR) targeting highly conserved regions of the HIV-1 genome (e.g., LTR or gag regions). Results are expressed as copy numbers per 10^6 CD4+ T cells or 10^6 PBMCs.

Intact Proviral DNA Assay (IPDA): high-molecular-weight genomic DNA is analyzed via multiplex digital droplet PCR (ddPCR). Dual-target probes independently amplify intact env and packaging signal (psi) regions. Droplets scoring positive for both targets reflect intact proviruses mostly capable of replication; droplets positive for a single target reflect 5'- or 3'-defective proviruses. Results are corrected for DNA shearing using a host gene reference assay (RPP30).

Systemic Inflammatory and Immune Activation Biomarkers: plasma samples will undergo targeted proteomic analysis using enzyme-linked immunosorbent assays (ELISA) or multiplex luminex panels. Biomarkers evaluated include Interleukin-6 (IL-6), soluble CD14 (sCD14), soluble CD163 (sCD163), IP-10 (CXCL10), and high-sensitivity C-reactive protein (hs-CRP).

Statistical Analysis Plan Primary Endpoint Analysis. Multivariable conditional logistic regression models will be constructed to evaluate the association between total HIV-DNA and intact proviral DNA levels (analyzed as continuous variables and across stratified quartiles) and the odds of HIV-RNA virological rebound. Odds ratios (OR) with 95% confidence intervals (CI) will be reported.

Adjustment Factors: regression models will adjust for key potential confounders, including baseline/nadir CD4+ T-cell count, pre-treatment HIV-RNA peak, HIV-1 subtype, initial anchor drug class (INSTI, NNRTI, or PI), duration of ART prior to baseline, and baseline inflammatory biomarker levels.

Receiver Operating Characteristic (ROC) Analysis: Area Under the Curve (AUC) metrics will be calculated to compare the diagnostic sensitivity, specificity, and positive/negative predictive values of total HIV-DNA versus IPDA intact proviral DNA for predicting virological rebound.

Correlation Analysis: non-parametric Spearman correlation coefficients will assess relationships between viral reservoir parameters (total DNA, intact DNA, defective DNA ratio) and plasma inflammatory marker concentrations.

Clinical Significance and Expected Impact. Validation of total HIV-DNA and intact proviral DNA as predictive biomarkers will provide clinicians with robust tools to identify PWH at elevated risk for virological failure despite apparent viral suppression. Incorporating reservoir profiling into routine clinical care will enable personalized medicine strategies, such as tailoring viral load monitoring intervals, guiding treatment optimization, and selecting optimal candidates for future HIV cure interventions and analytical treatment interruptions.