Mechanisms of HIV non-progression; robust and sustained CD4+ T-cell proliferative responses to p24 antigen correlate with control of viraemia and lack of disease progression after long-term transfusion-acquired HIV-1 infection

Dyer, Wayne B; Zaunders, John J; Yuan, Fang Fang; Wang, Bin; Learmont, Jennifer C; Geczy, Andrew F; Saksena, Nitin K; McPhee, Dale A; Gorry, Paul R; Sullivan, John S

DNA from PBMC was isolated using a QIAamp DNA mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. A nested polymerase chain reaction (PCR) was used to amplify ~1.5 kb of the HIV gag gene using the following primers: RNA was isolated from plasma using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Gag gene was amplified using the QIAGEN OneStep RT-PCR Kit using the outer primer pairs mentioned above. Second round PCR reactions were performed using the inner primer pair under the same conditions. PCR products were purified using a Millipore PCR purification plate (Millipore, Billerica, MA, USA) and sequenced by the ABI PRISM BigDye Terminator V3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI 377 automated sequencer. Multiple sequences derived from each patient were analysed using Sequencher 3.11 software (Gene Codes Corp., Ann Arbor, MI, USA). Chromatograms derived from both forward and reverse primers were aligned with the reference strain HIV-1 HXB2.