Capitalizing on Post-exposure ARV Prophylaxis to Restrict Seeding of the HIV Reservoir. PCR (ddPCR), across different HIV-1 subtypes. Study Design. The JZL184 analytical sensitivity and specificity of an HIV-1 DNA ddPCR assay was decided using serial dilutions of a plasmid made up of HIV-1 LTR-gag spiked JZL184 into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. Results. The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. Conclusions. This assay has applications for detecting occult HIV-1-contamination in the setting of combination and long-acting regimens utilized for HIV-1 prevention, across different HIV-1 subtypes, in infants and adults, and in HIV-1 remedy interventions. strong class=”kwd-title” Keywords: HIV-1, ddPCR, methods Background In HIV-1-uncovered infants more youthful than age 18 Mouse monoclonal to CD14.4AW4 reacts with CD14, a 53-55 kDa molecule. CD14 is a human high affinity cell-surface receptor for complexes of lipopolysaccharide (LPS-endotoxin) and serum LPS-binding protein (LPB). CD14 antigen has a strong presence on the surface of monocytes/macrophages, is weakly expressed on granulocytes, but not expressed by myeloid progenitor cells. CD14 functions as a receptor for endotoxin; when the monocytes become activated they release cytokines such as TNF, and up-regulate cell surface molecules including adhesion molecules.This clone is cross reactive with non-human primate months, HIV-1 antibody screening cannot distinguish between infant and maternally-derived antibodies to allow use for infant HIV-1 diagnosis (1, 2). Quantitative or qualitative real-time polymerase chain reaction (RT-PCR) assays that detect HIV-1 RNA in plasma or DNA in peripheral blood mononuclear cells (PBMCs) are therefore required (3, 4). Early antiretroviral treatment (ART) of perinatal HIV-1 contamination can lead to low concentrations of circulating HIV-1 infected cells in association with unfavorable HIV-1 antibody and DNA tests by clinical diagnostic assays (5C11), possibly leading to unnecessary ART interruption in later child years (12). Furthermore, the use of combination antiretroviral (ARV) prophylaxis in high-risk perinatal exposures, along with very early ART within days of age or directly transitioning from prophylaxis to ART, may lead to low infected cell concentrations (7, 13C16) that require more sensitive HIV-1 DNA assays to confirm infection on ART. HIV-1 DNA concentrations in peripheral blood are also used to determine therapeutic efficacy of remedy strategies aimed at eradicating reservoir cells in ART-free remission and remedy (17, 18). Sensitive and clinically validated HIV-1 DNA assays may become useful in these specialized settings, in both perinatal and adult infections. Objectives We optimized and clinically validated an HIV-1 DNA quantitative assay using the more sensitive and precise approach of droplet digital PCR (ddPCR), across different HIV-1 subtypes, with potential applications for use JZL184 clinically and in HIV-1 prevention and cure trials (19). Study Design A detailed description of the DNA isolation and ddPCR is usually provided in the supplemental materials. Preparation of cell pellets and plasmid requirements. PBMCs were isolated from leukopaks (New York City Blood Center, New York NY) using Ficoll-Hypaque centrifugation (GE Healthcare, Chicago IL), treated with Red Blood Cell Lysis Buffer (eBiosciences, San Diego CA) and aliquoted into 5-million cell pellets for storage at ?80 C until further use. Plasmid requirements. A plasmid made up of a pMK-RQ-Bs backbone (GeneArt Gene Synthesis Technology; Thermo Scientific, Waltham MA) with a 160-bp region of the HIV-1C5 LTR and gag region (positions 626 C 786, HXB2) (20), as well as a fragment of the housekeeping gene Ribonuclease Protein Subunit P30 (RPP30) (21), was constructed (Supplemental Physique 1). Prior to testing, an aliquot of plasmid stock was serially diluted and spiked into 5-million PBMC pellets to reach final concentrations ranging from 1,000 to 1 1.25 copies per 106 PBMCs. A second high-dilution series (10, 000 to 100 copies per 106 PBMCs) was tested to assess linearity at higher concentrations. DNA Isolation. Genomic DNA was isolated from your combined plasmid-cell mixtures using QIAamp DNA Blood Midi Kit (Qiagen, Germantown MD) with the addition of an overnight ethanol precipitation step to obtain highly concentrated and real genomic DNA for downstream ddPCR applications (21). Primers, Probes and PCR conditions. The primers and probes for HIV-1 LTR-gag (20) and RPP30 (21) (Supplemental Table 1) were obtained from Integrated DNA Technologies, Coralville IA), reconstituted in DNase/RNase-free water, and stored at concentrations of 18 M primers and 5 M probes in 50 L aliquots for single JZL184 use only. Genomic DNA preparation and ddPCR. Genomic DNA (sufficient to perform 8 replicates at 1000 ng input per well) was subjected to restriction digest with XbaI (New England BioLabs, Ipswich MA). Unfavorable control wells made up of genomic DNA without plasmid were performed in four replicates, along with a single control well without genomic DNA as a no template control (NTC). To directly quantify the number of cells analyzed based JZL184 on copies of RPP30, PBMC genomic DNA was diluted 1:10 (100 ng DNA per well) since RPP30 is present in high copy number at two copies per cell. This allowed the results to be expressed as HIV-1 DNA copies per cells.
