Differential decreases in various HIV DNA regions and HIV transcripts after ART initiation during chronic infection

HIV-transcribing cells persist on ART and likely contribute to inflammation as well as the viral rebound that occurs after stopping ART. However, it is unclear how ART start time and prolonged time on ART affect the clearance of cells expressing HIV transcripts that differ in their processivity and/or the presence of mutations. To investigate these questions, we measured levels of various HIV transcripts and their corresponding HIV DNA regions in longitudinal samples of peripheral CD4+ T cells obtained from 10 individuals during untreated chronic infection (T1) and up to four additional timepoints on suppressive ART (median years after ART start: T2 = 0.9; T3 = 2.9; T4 = 5.2; T5 = 7). Before ART, the pattern of HIV transcripts suggested blocks to elongation and completion, and only ~1% of intact proviruses were transcribing intact HIV RNA. After ART, we observed decreases in most HIV transcripts. Multiply spliced HIV RNA tended to decrease faster than initiated HIV RNA, and intact HIV RNA decreased faster than 5' defective HIV RNA. ART reduced completed but not elongated or Pol HIV RNA as normalized to HIV DNA. While most proviruses and HIV transcripts showed no further decline after T2, multiply spliced HIV RNA continued to decline through T3, and R-U5-pre-Gag HIV DNA continued to decline through T4, suggesting their clearance may be dictated by different or more sustained immune mechanisms. No further changes were observed after T4. These findings suggest the need for new therapies that can target the remaining proviruses/transcripts and/or modulate the expression of completed, multiply spliced, and intact HIV RNA.IMPORTANCEEven in ART-treated people living with HIV (PWH), expression of different viral products may contribute to immune activation, inflammation, organ damage, and reduced life expectancy. We quantified different types of HIV DNA and transcripts in people living with untreated chronic HIV and up to 4 timepoints over 6-10 years on ART. During the first year on ART, multiply spliced HIV RNA decreased faster than total HIV RNA, and intact HIV RNA decreased faster than defective RNA. Multiply spliced HIV RNA continued to decline over 1-3 years on ART, and proviruses containing the 5' end declined over years 3-5, suggesting differences in the immune clearance of various HIV transcripts and proviruses. While intact HIV RNA became undetectable, levels of incomplete or defective HIV transcripts reached an equilibrium after 3 years on ART, indicating the limits of the immune system and ART to reduce incomplete/defective HIV transcripts that may still contribute to immune activation.