Monitoring HIV Cell Reservoirs with "SUSHI"
The use of HIV proviral DNA assays to identify reservoirs and quantify changes with disease progression and therapeutic approaches has eclipsed alternative analyte measurements notably that of cell associated HIV transcriptional activity.
Simultaneous Ultrasensitive Subpopulation Staining/Hybridization In Situ (SUSHI): A new Application of an Established Technique to Monitor Cell Reservoirs Expressing Unspliced HIV-1 gag-pol mRNA in response to Highly Active Antiretroviral Therapy.
The use of HIV proviral DNA assays to identify reservoirs and quantify changes with disease progression and therapeutic approaches has eclipsed alternative analyte measurements notably that of cell associated HIV transcriptional activity. Data from recent studies demonstrate cell associated HIV transcriptional activity in peripheral blood mononuclear cells from 62% to 89% patients on HAART with plasma viral load > 50 copies HIV-1 RNA/mL (1, 2). Most assays that measure HIV transcriptionally active cells require extracted and purified total cell associated RNA followed by reverse-transcription, polymerase chain reaction (RT-PCR) (1-4).
This provides an overall account of cell associated HIV transcriptional activity from total tissue (blood and otherwise) unless cells are pre-sorted prior to RNA extraction as was previously reported (5).
An alternate assay combines cell immunophenotyping followed by in-situ hybridization for unspliced HIV-1 gag-pol mRNA (gag-pol) while maintaining cell morphology, cell markers and intracellular HIV nucleic acid molecules. The technique, Simultaneous Ultrasensitive Subpopulation Staining/Hybridization In Situ (SUSHI) was first reported in 1998 (6) and used to elucidate HIV transcriptional activity (gag-pol+) in peripheral blood mononuclear cells (6-8), placenta (9, 10) and in an in-vitro organ culture model to study transmission of HIV-1 in the female genital tract (11). SUSHI was then evaluated in a small proof of concept study to measure changes in gag-pol+ reservoirs from a few patients on HAART. Depending on treatment outcome, results demonstrate varying correlations (from high to low and inverse) between gag-pol+ reservoirs and plasma viral load and between different reservoirs of the same or different cell lineage, a slower decline in gag-pol+ reservoirs compared to plasma viral load in the treatment success patient and an increase in gag-pol+ reservoirs several weeks prior to plasma viral load breakthrough in the treatment failure patient as similarly reported by Pasternak et al. (12). Cell populations studied included peripheral blood derived monocyte/macrophages, macrophages, total CD4+ cells, and naïve, memory and activated memory cells. Relative proportions of gag-pol+ reservoirs were in line with the literature; the more mature the greater the gag-pol+ reservoir; immature cells (naïve T cells and monocyte/macrophages) gag-pol reservoirs were either undetectable or slightly above the assay’s limit of detection. Another interesting finding was the altered morphology (increased size) of gag-pol+ cells and in order to capture these cells a broad light scatter was used to identify the immunophenotypic cell properties rather than normal lymphocyte gates that can miss a proportion of these gag-pol+ cells. Data from this proof of concept study are being prepared in a manuscript for submission. On another note, data from drug naïve patients show a staggering variation in the proportion of reservoirs both within and between patients; this possibly suggests a complex compartmentalization of HIV within peripheral blood cells; other tissues will need to be studied. Clinical trials have recently implemented SUSHI to further elucidate the dynamics of various PBMC derived reservoirs during HAART and results forthcoming.
The SUSHI technology is patented and used in the Virotect HIV-1 Viral Reservoir Assay (HIVVR: IncellDx Inc., Menlo Park, California).
More information can be obtained directly from Keith Shults, Director of Research, IncellDx, Phone: +1 (615) 294-5231, email:
Pr Tarek Elbeik short bio:
-Present: CEO, Elbeik Associates, LLC
-2004 – 2005: Professor in Research Virology, Laboratory Medicine, University of California, San Francisco
-1997 – 2005: Director, Microbiology Research Laboratory, Laboratory Medicine, University of California, San Francisco -1997 – 2004: Associate Researcher, Laboratory Medicine, University of California, San Francisco
-1988 – 1997: Co-Director, Virology Research Laboratory, Medicine, University of California, San Francisco
-1988 – 1997: Assistant Researcher, Medicine, University of California, San Francisco
-1987 – 1988: Post Doctoral Fellow, Laboratory Medicine, University of California, San Francisco
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Key words: HIV reservoirs