Low unspliced cell-associated HIV RNA in early treated adolescents living with HIV on long suppressive ART

Abstract

Introduction: Initiation of antiretroviral treatment (ART) in patients early after HIV-infection and long-term suppression leads to low or undetectable levels of HIV RNA and cell-associated (CA) HIV DNA and RNA. Both CA-DNA and CA-RNA, overestimate the size of the HIV reservoir but CA-RNA as well as p24/cell-free viral RNA can be indicators of residual viral replication. This study describes HIV RNA amounts and levels of cytokines/soluble markers in 40 well-suppressed adolescents who initiated ART early in life and investigated which viral markers may be informative as endpoints in cure clinical trials within this population.

Methods: Forty adolescents perinatally infected with HIV on suppressive ART for >5 years were enrolled in the CARMA study. HIV DNA and total or unspliced CA-RNA in PBMCs were analyzed by qPCR/RT-qPCR and dPCR/RT-dPCR. Cell-free HIV was determined using an ultrasensitive viral load (US-VL) assay. Plasma markers and p24 were analyzed by digital ELISA and correlations between total and unspliced HIV RNA and clinical markers, including age at ART, Western Blot score, levels of cytokines/inflammation markers or HIV CA-DNA, were tested.

Results: CA-RNA was detected in two thirds of the participants and was comparable in RT-qPCR and RT-dPCR. Adolescents with undetectable CA-RNA showed significantly lower HIV DNA compared to individuals with detectable CA-RNA. Undetectable unspliced CA-RNA was positively associated with age at ART initiation and Western Blot score. We found that a higher concentration of TNF-α was predictive of higher CA-DNA and CA-RNA. Other clinical characteristics like US-VL, time to suppression, or percent CD4+ T-lymphocytes were not predictive of the CA-RNA in this cross-sectional study.

Conclusions: Low CA-DNA after long-term suppressive ART is associated with lower CA-RNA, in concordance with other reports. Patients with low CA-RNA levels in combination with low CA-DNA and low Western Blot scores should be further investigated to characterize candidates for treatment interruption trials. Unspliced CA-RNA warrants further investigation as a marker that can be prioritized in paediatric clinical trials where the sample volume can be a significant limitation.

Keywords: HIV-1; adolescents; cell-associated DNA; cell-associated RNA; cytokines; early treated; suppressed.

Figure 1

Analysis of HIV-1 cell-associated nucleic acids in children on ART; (A) LTR- and pol-primer/probe binding sites in the HIV-1 genome; (B) Detection of HIV-1 DNA, total and unspliced CA-RNA in PBMCs; (C) Level of CA-DNA in children with undetectable (brown column) and detectable (green columns) total and unspliced CA-RNA; Wilcoxon matched-pairs signed rank test *p<0.05, ***p<0.01.

Figure 2

Detection of ultra-sensitive VL in CARMA participants. Samples with detectable CA-RNA plus CA-DNA are black, samples with negative CA-RNA are depicted with a green circle and samples with negative CA-DNA have a red center. Double-negative samples are red with a green circle. Shown is the mean of all samples with 95% confidence interval.

Figure 3

Clinical markers in groups of participants with detectable and undetectable CA-RNA; (A) Age at ART, (B) Baseline VL, (C) Time to suppression, (D) %CD4 T-lymphocytes, (E) %CD8 T-lymphocytes, (F) Western Blot Score. Shown are medians with 95% confidence interval; Wilcoxon matched-pairs signed rank test *p<0.05, ***p<0.01.

Figure 4

Importance of different cytokines and inflammation markers for prediction of increasing (A) HIV CA-DNA; (B) total CA-RNA; (C) unspliced CA-RNA.