Open AccessDevelopment of sensitive dd PCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms
Author(s) -
Bosman Kobus J,
Wensing Annemarie MJ,
Pijning Aster E,
Snippenberg Wilco J,
Ham Petra M,
Jong Dorien MC,
Hoepelman Andy IM,
Nijhuis Monique
Publication year - 2018
Publication title -
journal of the international aids society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.724
H-Index - 62
ISSN - 1758-2652
DOI - 10.1002/jia2.25185
Subject(s) - plasmid , recombinant dna , virology , digital polymerase chain reaction , biology , polymerase chain reaction , group specific antigen , long terminal repeat , dna , microbiology and biotechnology , medicine , human immunodeficiency virus (hiv) , gene , genome , genetics
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 QX 200), 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 ( CRF s) 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 CRF s (A, B, C, D, AE , F, G and AG ), justifying their application in future trials aimed at global HIV cure.