Amplicor version 1

We quantified HIV-1 RNA from plasma using the Roche Amplicor Version 1.5 assay (Rotkreuz, Switzerland), and measured CD4+ T cell counts by flow cytometry. These assays were done on location by the clinical centres recruiting the patients, using fresh plasma and cells.

The slow progressor cohort of HIV-infected children has been previously described [15 (link)]. Informed consent was provided for participation of the subjects in the study. Ethics approval was given by the University of the Free State Ethics Committee, Bloemfontein, the Biomedical research Ethics Committee, University of KwaZulu-Natal, Durban, and the Oxford Research Ethics Committee. Viral load in chronic infection was measured using the Roche Amplicor version 1.5 assay; CD4⁺ T cell counts were measured by flow cytometry.
Four-digit HLA typing of the Class I locus was performed from genomic DNA as previously described [20 (link)] by sequence-based typing at the ASHI^* accredited HLA typing laboratory, University of Oklahoma Health Sciences Centre, USA. Exons 2 and 3 of HLA Class I were amplified by locus-specific PCR and then sequenced. Resolution of ambiguities was undertaken according to the ASHI committee recommendations.
Additional viral sequence analyses were performed on a previously described, multi-center cohorts: 1470 African clade C Gag sequences from cohorts based in Durban [21 (link)], Bloemfontein [22 (link)] and Kimberley [23 (link)] South Africa, Zambia and the Thames Valley area of the United Kingdom [23 (link)].

Study subjects were chosen from the CHARTER cohort. De-identified, contemporaneous, cryopreserved CSF and plasma samples from fifteen chronically infected, HIV-1 seropositive individuals with normal neurocognitive performance (NCN), Asymptomatic Neurocognitive Impairment (ANI) and Mild Neurocognitive Disorder (MND) were provided.
Neurocognitive testing and clinical histories were obtained at CHARTER study visits by trained psychometrists and research staff. Participants underwent a comprehensive neurocognitive battery of tests within seven cognitive domains: speed of information processing, learning, recall, abstraction/executive functioning, verbal fluency, attention/working memory and motor skills. Following the demographic correction of T-scores for each test measure, a global deficit score (GDS), based on number and magnitude of impaired test performances was determined. At the time of neurocognitive testing, contemporaneous cerebrospinal fluid (CSF) and peripheral-blood samples were obtained from each individual by lumbar puncture and routine phlebotomy. Peripheral blood CD4+ T cell counts were performed at CHARTER research sites using routine established methods. HIV-1 RNA levels in the CSF and plasma were determined using the Roche Amplicor, version 1.5, with a lower limit of quantitation of 50 copies/mL.

Specimens from adult subjects from the following cohorts were used in our study:
HIV plasma viral load was measured by the Roche Amplicor version 1.5 assay with with COBAS AmpliPrep or TaqMan 48 for the Acute HIV Infection cohort or with COBAS Amplicor for the other cohorts. CD4+ T-cell counts were determined by flow cytometry using standard clinical protocol. CD4+ T-cell counts and viral loads were measured on clinical grounds and the data were supplied by the Centre of Recruitment.HLA typing was performed using a locus specific PCR amplification strategy and a heterozygous DNA sequencing methodology for exon 2 and 3 of the class I PCR amplicon [21 (link)].

We studied 257 HIV-infected ART-naive adult and paediatric subjects recruited via three previously described cohorts (Table 1):
In total, we used 62 paediatric (all from the Kimberley cohort) and 195 adult subject samples for ELISpot studies. We used an additional 37 subject samples from Durban cohort for NW8-tetramer staining. The samples were collected between 2009–2014. HIV viral load was measured using the Roche Amplicor version 1.5 assay according the manufacturer’s instructions; CD4+ T-cell counts were measured by flow cytometry. The CD4% is calculated as the percentage of live CD3+ CD4+ T-cells in total lymphocytes. The CD4% percentages in children were recorded at the recruitment site.