Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

被引:19
作者
Bosman, Kobus J. [1 ]
Wensing, Annemarie M. J. [1 ]
Pijning, Aster E. [1 ]
van Snippenberg, Wilco J. [1 ]
van Ham, Petra M. [1 ]
de Jong, Dorien M. C. [1 ]
Hoepelman, Andy I. M. [2 ]
Nijhuis, Monique [1 ]
机构
[1] Univ Med Ctr Utrecht, Dept Med Microbiol, Heidelberglaan 100, NL-3508 GA Utrecht, Netherlands
[2] Univ Med Ctr Utrecht, Dept Internal Med & Infect Dis, Utrecht, Netherlands
关键词
human immunodeficiency virus; subtypes; reservoir; cure; quantification; digital PCR; IMMUNODEFICIENCY-VIRUS TYPE-1; DROPLET DIGITAL PCR; SUPPRESSIVE ANTIRETROVIRAL THERAPY; BROADLY NEUTRALIZING ANTIBODIES; STEM-CELL TRANSPLANTATION; POLYMERASE-CHAIN-REACTION; REAL-TIME PCR; ACCURATE QUANTIFICATION; HIV-1-INFECTED PATIENTS; PERIPHERAL-BLOOD;
D O I
10.1002/jia2.25185
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
Introduction: The latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype-tolerance using digital PCR. Methods: A subtype-B-specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype-tolerance in digital PCR (Bio-Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide. Results: HIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R-2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R-2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty-nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG. Conclusions: The results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non-B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.
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页数:13
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共 102 条
  • [1] Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots
    Aitken, Susan C.
    Kliphuis, Aletta
    Bronze, Michelle
    Wallis, Carole L.
    Kityo, Cissy
    Balinda, Sheila
    Stevens, Wendy
    Spieker, Nicole
    de Oliveira, Tulio
    de Wit, Tobias F. Rinke
    Schuurman, Rob
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2013, 51 (06) : 1899 - 1905
  • [2] Rational design of HIV-1 fluorescent hydrolysis probes considering phylogenetic variation and probe performance
    Althaus, Claudia F.
    Gianellaa, Sara
    Rieder, Philip
    von Wyl, Viktor
    Kouyos, Roger D.
    Niederoest, Barbara
    Schmid, Adrian
    Metzner, Karin J.
    Joos, Beda
    Guenthard, Huldrych F.
    Fischer, Marek
    [J]. JOURNAL OF VIROLOGICAL METHODS, 2010, 165 (02) : 151 - 160
  • [3] Is HIV-1 evolving to a less virulent form in humans?
    Arien, Kevin K.
    Vanham, Guido
    Arts, Eric J.
    [J]. NATURE REVIEWS MICROBIOLOGY, 2007, 5 (02) : 141 - 151
  • [4] Armbruster David A, 2008, Clin Biochem Rev, V29 Suppl 1, pS49
  • [5] LTR Real-Time PCR for HIV-1 DNA Quantitation in Blood Cells for Early Diagnosis in Infants Born to Seropositive Mothers Treated in HAART Area (ANRS CO 01)
    Avettand-Fenol, Veronique
    Chaix, Marie-Laure
    Blanche, Stephane
    Burgard, Marianne
    Floch, Corinne
    Toure, Kadidia
    Allemon, Marie-Christine
    Warszawski, Josiane
    Rouzioux, Christine
    [J]. JOURNAL OF MEDICAL VIROLOGY, 2009, 81 (02) : 217 - 223
  • [6] Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters
    Benki, Sarah
    McClelland, R. Scott
    Emery, Sandra
    Baeten, Jared M.
    Richardson, Barbra A.
    Lavreys, Ludo
    Mandaliya, Kishorchandra
    Overbaugh, Julie
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 2006, 44 (12) : 4357 - 4362
  • [7] AN ANTIBODY THAT BINDS THE IMMUNOGLOBULIN CDR3-LIKE REGION OF THE CD4 MOLECULE INHIBITS PROVIRUS TRANSCRIPTION IN HIV-INFECTED T-CELLS
    BENKIRANE, M
    CORBEAU, P
    HOUSSET, V
    DEVAUX, C
    [J]. EMBO JOURNAL, 1993, 12 (13) : 4909 - 4921
  • [8] Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems
    Bialek, Julia K.
    Dunay, Gabor A.
    Voges, Maike
    Schaefer, Carola
    Spohn, Michael
    Stucka, Rolf
    Hauber, Joachim
    Lange, Ulrike C.
    [J]. PLOS ONE, 2016, 11 (06):
  • [9] Human Immunodeficiency Virus Type 1 Monoclonal Antibodies Suppress Acute Simian-Human Immunodeficiency Virus Viremia and Limit Seeding of Cell-Associated Viral Reservoirs
    Bolton, Diane L.
    Pegu, Amarendra
    Wang, Keyun
    McGinnis, Kathleen
    Nason, Martha
    Foulds, Kathryn
    Letukas, Valerie
    Schmidt, Stephen D.
    Chen, Xuejun
    Todd, John Paul
    Lifson, Jeffrey D.
    Rao, Srinivas
    Michael, Nelson L.
    Robb, Merlin L.
    Mascola, John R.
    Koup, Richard A.
    [J]. JOURNAL OF VIROLOGY, 2016, 90 (03) : 1321 - 1332
  • [10] RAPID AND SIMPLE METHOD FOR PURIFICATION OF NUCLEIC-ACIDS
    BOOM, R
    SOL, CJA
    SALIMANS, MMM
    JANSEN, CL
    WERTHEIMVANDILLEN, PME
    VANDERNOORDAA, J
    [J]. JOURNAL OF CLINICAL MICROBIOLOGY, 1990, 28 (03) : 495 - 503