Fig. 2: HIV-RNA persistence in lymph nodes of Fiebig I/II treated individuals.

HIV-RNA detection in lymph nodes (LN) of Fiebig I/II treated individuals using RNAscope (a–e) and Cobas^® AmpliPrep HIV-1 test (f–i). a RNAscope hybridization for HIV gag-pol RNA was detected using 3, 3’-diaminobenzidene (DAB, brown) or b, c fluorescent Opal polymers (green). Representative images for HIV negative, Fiebig I/II treated (Tx), late Tx and untreated (unTx) HIV-infected LN sections are shown. Single RNA transcripts are seen as punctate dots; clusters of transcripts are also observed. Red arrowheads identify HIV RNA⁺ cells and green arrowheads identify virions on follicular dendritic cells. c Images showing multiplexed RNAscope gag-pol hybridization (green) coupled with IF staining for CD4⁺ cells (red). Three independent experiments were conducted with similar results. Scale bars are 50 μm (a) or 20 μm (b, c). d RNA signals quantified in micrographs using Fiji [(ImageJ software, Fiebig I/II Tx, n = 12; Fiebig III–V Tx, n = 4; late Tx, n = 2; and unTx, n = 4). Five fields of view are analyzed per sample and averaged. e A correlation analysis of area staining of gag-pol RNA and Gag p24 density for Fiebig I/II Tx LNs. f Viral RNA loads are quantified in lymph node mononuclear cells (LNMCs) (Fiebig I/II Tx, n = 7; Fiebig III–V Tx, n = 4; late Tx, n = 9; and unTx, n = 7). Viral loads below the limits of detection of the assay are assigned a value of 20. Correlation analysis of LNMCs’ viral loads with g peak plasma viral loads, h treatment duration, and i time to suppression for Fiebig I/II Tx, n = 7; and Fiebig III–V, n = 4; donors. All statistical tests are two-sided and p values are from the Mann–Whitney U test (d, f). Spearman rho (r) values and p values are reported for correlation analyses (e, g–i). Dotted line denotes threshold of viral load detection. Error bars represent interquartile range (d, f). Data are presented as median and interquartile range (d, f). Source data are provided as a source data file.