New Tool for HIV Reservoirs PDF Print E-mail
Written by Tarek Elbeik   
Saturday, 15 October 2011 14:48

Monitoring HIV Cell Reservoirs with "SUSHI"

testtubesThe 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: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

 

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


References:


1. Kupfer B, Matz B, Däumer MP, Roden F, Rockstroh JK, Qurishi N, Spengler U, Kaiser R. Frequent detection of cell-associated HIV-1 RNA in patients with plasma viral load <50 copies/ml. J Med Virol. 2007 Oct;79(10):1440-5.

2. Pasternak AO, Adema KW, Bakker M, Jurriaans S, Berkhout B, Cornelissen M, Lukashov VV. Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA. J Clin Microbiol. 2008 Jul;46(7):2206-11. Epub 2008 May 7.

3. Gil C, Garcia MT, Garcia F, Miró JM, Agüero F, Alós L, Zamora L, Capón A, Costa J, Pumarola T, Gatell JM. Evaluation of the Roche COBAS(®) TaqMan(®) HIV-1 test for quantifying HIV-1 RNA in infected cells and lymphoid tissue. J Virol Methods. 2011 Mar 31.

4. Soares RS, Tendeiro R, Foxall RB, Baptista AP, Cavaleiro R, Gomes P, Camacho R, Valadas E, Doroana M, Lucas M, Antunes F, Victorino RM, Sousa AE Cell-associated viral burden provides evidence of ongoing viral replication in aviremic HIV-2-infected patients.  J Virol. 2011 Mar;85(5):2429-38.

5. Kaiser P, Joos B, Niederöst B, Weber R, Günthard HF, Fischer M. Productive human immunodeficiency virus type 1 infection in peripheral blood predominantly takes place in CD4/CD8 double-negative T lymphocytes. J Virol. 2007 Sep;81(18):9693-706. Epub 2007 Jul 3.

6. Patterson BK, Mosiman VL, Cantarero L, Furtado M, Bhattacharya M, Goolsby C. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype. Cytometry. 1998 Apr 1;31(4):265-74.

7. Patterson BK. Simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI) in HIV-1 disease monitoring. Methods Mol Bio 2010;659,337-46.

8. Pett SL, McCarthy MC, Cooper DA, MacRae K, Tendolkar A, Norris R, et al. A phase I study to explore the activity and safety of SCH532706, a small molecule chemokine receptor-5 antagonist in HIV type-1-infected patients. Antivir Ther 2009;14,111-15.

9. Patterson BK, Behbahani H, Kabat WJ, Sullivan Y, O'Gorman MR, Landay A, et al. Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women. J Clin Inves 2001;107,287-94.

10. Behbahani H, Popek E, Garcia P, Andersson J, Spetz AL, Landay A, et al. Up-regulation of CCR5 expression in the placenta is associated with human immunodeficiency virus-1 vertical transmission. Am J Pathol 2000;157,1811-18.

11. Collins KB, Patterson BK, Naus GJ, Landers DV, Gupta P. Development of an in vitro organ culture model to study transmission of HIV-1 in the female genital tract. Nat Med 2000;6,475-79.

12. Pasternak AO, Jurriaans S, Bakker M, Prins JM, Berkhout B, Lukashov VV. Cellular levels of HIV unspliced RNA from patients on combination antiretroviral therapy with undetectable plasma viremia predict the therapy outcome. PLoS One. 2009 Dec 31;4(12):e8490.



 


Key words: HIV reservoirs
Last Updated on Sunday, 16 October 2011 11:13
 

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QIng Liu 16.10.2011 (18:48:34)  
SUSHI assay for HIV Reservoir Yes No  

FYI. This is a new assay for HIV reservoir and we might want to use it in the future. JAZ

 
   
       

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