Treatment-induced decline of human immunodeficiency virus-1 p24 and HIV-1 RNA in lymphoid tissue of patients with early human immunodeficiency virus-1 infection

Abstract

We report detailed quantitative analysis of human immunodeficiency virus-1 (HIV-1) p24 and HIV-1 RNA in tonsil biopsies from 13 patients with early, asymptomatic HIV infection before and during combination antiretroviral therapy. Using fluorescent microscopy in conjunction with reverse transcriptase-polymerase chain reaction of frozen tissue sections, we show that plasma and tissue viral loads decreased by approximately 3 logs during the 1-year treatment period, with good correlation between the HIV-1 p24 and HIV-1 RNA response in tissue. The decrease of tissue viral load was delayed compared to plasma viral load, possibly explained by the observation that the amount of follicular dendritic cell-associated virus correlated best with the area under the curve of plasma HIV-1 RNA throughout the last 12 weeks. Before and during treatment, the relative proportions of HIV-1 on follicular dendritic cells and within mononuclear cells remained constant, suggesting similar decay characteristics in these two lymphoid tissue compartments. However, viral p24 or RNA remained almost always detectable in tissue despite full suppression of HIV-1 RNA in plasma, and increased even after short-term rebounds in plasma viral load. Thus, full and sustained suppression of viral replication was required to efficiently decrease viral load in lymphoid tissue, but complete abolition of residual viral replication was not achieved.

Figure 1.

ISH detection of HIV-1 RNA in a tonsil frozen section. Gray scale images display the follicular distribution of HIV-1 RNA (Cy3 fluorescence) (b) and an infected cell from a different field (b, inset); cell nuclei in the same fields are revealed by DAPI fluorescence (c). d: An overlay of b (colored red) and c (colored blue), shows the location of HIV-1 RNA relative to the cell nuclei. The corresponding H&E-stained field from a neighboring section is shown in a. Scale bars, 50 μm.

Figure 2.

IHC detection of HIV-1 p24 and cellular markers. a: Color overlay (blue, nuclei; red, p24; green, FDC; red/green combinations appear as orange to yellow) shows an entire section composed of 25 montaged fields (original magnification, ×10). b–g: Show the same high-power field of a lymphoid follicle (original magnification, ×40; scale bar in b, 50 μm). b–d: Black and white images of the p24 (Cy3), FDC (Cy5), and T cell (Alexa488) fluorescent channels. e–f: Color overlays, as follows: overlay of nuclei (blue, DAPI) and p24 (red; e); nearly complete co-localization (orange to yellow) of p24 (red) and FDC (green) within the follicle, whereas the infected cell (arrow) does not co-localize with FDC (f); co-localization of the infected cell with CD3 (yellow), no co-localization of CD3 (green) with follicular p24 (red) (g).

Figure 3.

Co-localization of p24 and cell marker fluorescence. Total p24 Fl (circle; average of 41 p24-positive biopsies) was subdivided into intracellular (black), extracellular (Xcell) FDC-associated (vertical lines), extracellular CD3-associated (horizontal lines), extracellular FDC and CD3-associated (cross-hatch pattern), and extracellular p24 not otherwise colocalized (white). The FI derived from each subset is indicated as percent of total p24 FI.

Figure 4.

Co-localized subsets of HIV p24 from one field (original magnification, ×40; scale bar, 25 μm). a–d: Black and white images of each fluorescent channel: nuclei (DAPI) (a), HIV p24 (Cy3) (b), FDC (Cy5) (c), and T cells (CD3, Alexa488) (d). e: Nuclei (blue) overlaid with binary masks showing distributions of FDC (red), CD3 (green), or both markers in the same pixel (yellow). f: Distribution of p24 (red) relative to cell nuclei (blue). g: HIV p24 signal is color encoded according to co-localization with cell markers (color intensity is proportional to fluorescent intensity); p24 + FDC (red), p24 + CD3 (green), p24 + FDC + CD3 (yellow), p24 not colocalized (gray). h: For comparison, this contrast-enhanced copy of g shows color encoding more clearly.

Figure 5.

HIV-1 RNA ISH expressed as fluorescent granules per section (left) and p24 ICC fluorescent intensity (Fl) per section (right) compared with HIV-1 RNA copies/section. Solid lines show expected theoretical relationship based on the formulas shown, and dotted lines indicate a range of ±1 log₁₀ (see text for details).

Figure 6.

Decreases from baseline in HIV-1 RNA (VL) in plasma, and HIV-1 RNA (VL) and p24 in lymphoid tissue; in all patients (top), and in those without (group A) and with (group B) viral rebound during treatment. Box and whiskers show median, 25th, and 75th percentile and range of the data. n.s., not significant; VL, viral load.

Figure 7.

Correlations of decreases from baseline to week 4 (left) and 48 (right) in HIV-1 RNA (VL) in plasma, and HIV-1 RNA (VL) and p24 in lymphoid tissue. For calculations of log₁₀ decreases, measurements below the detection limit were assigned the lower limit of detection (shown with solid data points or marked with X; see legend inserts).

Figure 8.

HIV-1 RNA (VL) and p24 in lymphoid tissue correlate better with area under the curve of HIV-1 RNA (VL) in plasma throughout the last 12 weeks before biopsy (top) than with the actual plasma HIV-1 RNA (VL) at the time of biopsy (bottom). Solid data points represent samples with undetectable tonsil p24 (left) or tonsil VL (right) at the corresponding detection limit, measurements from time points with undetectable plasma VL are marked with X. GV, gray value (fluorescent intensity); wk, week; VL, viral load.