Identification, Localization, and Quantification of HIV Reservoirs Using Microscopy

Abstract

The major barrier to eradicating human immunodeficiency virus-1 (HIV) infection is the generation and extended survival of HIV reservoirs. In order to eradicate HIV infection, it is essential to detect, quantify, and characterize circulating and tissue-associated viral reservoirs in infected individuals. Currently, PCR-based technologies and Quantitative Viral Outgrowth Assays (Q-VOA) are the gold standards to detect viral reservoirs. However, these methods are limited to detecting circulating viral reservoirs, and it has been shown that they misrepresent the size of the reservoirs, largely because they detect only one component of the HIV life cycle and are unable to detect viral reservoirs in tissues. Here, we described the use of multiple detection systems to identify integrated HIV DNA or viral mRNA and several HIV proteins in circulating and tissue reservoirs using improved staining and microscopy techniques. We believe that this imaging-based approach for detecting HIV reservoirs will lead to breakthroughs necessary to eradicate these reservoirs. © 2018 by John Wiley & Sons, Inc.

Keywords: AIDS; Q-VOA; T cells; astrocytes; macrophages; transmission.

Figure 1

Detection of HIV Nef DNA and HIV-p24 protein in PBMCs smears obtained from HIV infected individuals with no viral replication detected by at least 5 years. Six representative images of areas with HIV Nef DNA from six different individuals with undetectable replication. Using the technique described, we are able to detect a single copy of HIV integrated DNA (arrows) in non- replicating CD4⁺ T lymphocytes as determined by ELISA and PCR (>20 copies/ml). To demonstrate that these sequences of integrated DNA are productive, we stained for HIV-p24 (arrowheads). The merged picture shows the colocalization of nuclei (DAPI, blue staining to quantify a total number of cells), HIV DNA (Red nuclear staining), and HIV protein (HIV-p24, green staining). Note that not all cells with inserted DNA produce HIV-p24 protein. Note that in patient 2, we are unable to detect any HIV Nef DNA despite that we analyzed 1.2×10⁷ cells. No positive signal was detected in uninfected individuals with HIV-DNA or HIV-p24 protein. Each picture corresponds just to the positive cells in thousands of negative fields. Most of the circulating cells with HIV Nef DNA were CD4 positive cells. Arrows denote cells with integrated HIV Nef DNA and the arrowhead indicates placed where HIV-p24 is accumulated.

Figure 2

Detection of HIV Nef DNA, Alu repeats (host DNA), nucleus (DNA) and a macrophage marker (Iba-1) in primary cultures of human macrophages after 2 days post-HIV infection. Using the described technique, we are able to detect the first and maybe the second cycle of HIV replication in these primary cells. Similar results were found using viral reservoirs, latently infected T cells and macrophages. The pictures show the staining of human primary cultures of macrophages for nuclei (DAPI, blue staining to quantify a total number of cells), HIV Nef DNA (green nuclear staining), and a macrophage marker (red staining, Iba-1) in uninfected and HIV infected conditions. Note the perfect colocalization of the Alu repeat and Nef probe as well as DAPI in the HIV infected cultures. This point is essential to demonstrate insertion of HIV DNA into the host DNA.

Figure 3

Detection of HIV Nef DNA in human vaginal tissue obtained from HIV infected individuals in ART for at least 15 years. Using the described techniques, we are able to detect few HIV infected cells in clinically suppressed individuals. Staining corresponds to nuclear (DAPI, blue staining), a macrophage marker (Iba-1, green staining), HIV Nef DNA (HIV Nef DNA, red staining), and Alu repeats (Alu sequences, white staining). Merge of all colors and phase contrast is also shown. Arrow represents a single positive area in a total area of 0.5 mm² of the biopsy. Clearly, the staining shows a population of HIV infected macrophages and T cells that are positive for the virus. Thus, this technique allows the identification and quantification of HIV reservoirs in multiple tissues even in patient samples without reactivation.