Detecting Sources of Immune Activation and Viral Rebound in HIV Infection

Abstract

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA⁺/p24⁺ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Keywords: HIV; immune activation; viral rebound.

FIG 1

RNAscope ISH/ELF 97 detection of HIV-producing cells in 5 μm sections of fixed and embedded induced ACH-2 cells. HIV virions appear green in the image. Nuclei were counterstained red with TOTO 3. In this 2D image, the largest number of virions are associated with a whole cell in the focal plane of the z-series. Scale bar = 10 μm.

FIG 2

Logarithmic plot of the average of replicates of induced ACH-2 cell equivalents and mapped HIV RNA reads from the EDITS assay results in Table 2. The values for the 2,500-cell point are shown as the isolated red data point, which is outside the linear range of the assay. The fitted line shows the linear relationship up to 320 cells (Pearson’s r = 0.99734).

FIG 3

(A) Flow cytometry dot plots showing the frequency of induced ACH-2 cells, serially diluted in CEM cells and measured by HIV-Flow. The expected frequencies are indicated at the top of each dot plot. The p24⁺ cells are depicted in red, and the p24⁻ cells are depicted in gray. (B) Correlation between HIV-flow estimates of p24⁺ cells per million cells and the expected frequency in the serial dilution series.

FIG 4

Flow cytometry example plots of the HIV RNA Flow-FISH assay and the correlation between measured and expected frequencies of infected cells in spiking experiments. (A) The p24⁺ gag mRNA⁺ ACH-2 cells before and after induction with PMA/ionomycin. (B) Highest frequency of reactivated ACH-2 spiked into the CEMx174 line. (C) False positive event rate in pure CEMx174 cells. (D) Theoretical expected frequency of 1.5 to 1,500 events per million cells and experimental frequencies of HIV⁺ events experimentally identified by HIV mRNA/protein co-staining.

FIG 5

Comparison of EDITS and IPDA assays for identifying HIV proviruses with deletions. (A) Schematic of HIV provirus and env mRNA. Red lines indicate the positions of the EDITS assay primers. (B) Comparison of the assays to detect deletions in proviruses a-f. The EDITS primers and amplicons are displayed in red, and the IPDA primers and amplicons are displayed in black.