Supplementary methods Sequencing of Selected Gag Region:
DNA was extracted from PBMCs collected at week 2 or 2.5 using the Qiagen DNeasy Blood and Tissue kit (Hilden, Germany) with an extended overnight proteinase K digestion incubation at 568C. Thegagregion targeted by the total HIV DNA assay was amplified for sequencing using the primers specified in supplementary table 1 (Ou CY, Science, 1988). Briefly, for a 50ml reaction, 1x buffer was used with 1.5 mM MgCl2, 200 nM dNTPs, 0.2ml of Taq, 400 nM of sense primer and 200 nM of antisense primer. Cycling conditions were 1 cycle of 948C for 2 minutes followed by 45 cycles of 948C for 30 seconds, 628C for 30 seconds and 72 for 30 seconds, and a further extension step of 728C for 7 minutes for 1 cycle. DNA was cleaned using the Qiagen QIAquick PCR purification kit protocol (Hilden, Germany).
Samples were sequenced at the ACRF facility at the Garvan Institute (Sydney, Australia) using the Big Dye Terminator 3.1 reaction. Results were analysed using MacVector Assembler Version 11 (North Carolina, USA).
DNA Real-Time PCR Analyses:
All real-time PCR assay results were normalized using the Applied Biosystems (ABI, Carlsbad, USA) TaqMan beta-actin detection reagents with a FAM-labeled probe. For the beta-actin assay, 12.5ml of iQ Supermix (Bio-Rad Laboratories California, USA) was used in a 25ml reaction, with 180 nM of forward and reverse primers and 120 nM of probe. The genomic DNA standard curve was constructed from purified human HIV negative buffy coat DNA. All assays were run using the Bio-Rad iQ supermix on a Bio-Rad iQ-5 multicolour real-time PCR detection system (BioRad, Hercules, USA). PCR conditions were as described for each assay. The limit of detection for all three HIV-1 DNA assays was 10 copies. All standard dilutions and patient samples were run in duplicate and HIV-1 DNA was expressed as copies/500ng DNA and further converted to copies/106cells.
Total HIV DNA Quantification:
Total DNA was extracted from CD4þT cell DCPs for weeks 0, 1, 2, 3, 4, 8, 12, 20, 24, 32 and 52 and PBMC for weeks 0, 4 and 52. HIV-1 DNA was quantified by a real-time PCR assay specific for HIV-gagas previously described (Suzuki K, Journal of RNAi and Gene Silencing 2005), using a pNL4-3 standard curve and primers SK145 and SKCC1B at a 800 nM concentration. The assay incorporated a fluorescent locked nucleic acid (LNA) probe SKLNA2-3 (supplementary table 1) at a concentration of 200 nM. PCR conditions consisted of 1 cycle of 958C for 3 minutes followed by 40 cycles of 958C for 15 seconds and 608C for 1 minute. The coefficient of variation (CV) for this assay was of 11%.
Patient-Specific Total HIV DNA Quantification:
The total HIV DNA assay was redesigned for one participant due to mismatches in the primers and probe. The sequences used are specified in supplementary table 1. All concentrations remained unchanged and the amplification temperature was increased to 64.58C. The standard curve used was a TOPO cloned patient insert.
Episomal 2-LTR HIV DNA Quantification:
Episomal 2-LTR HIV DNA was quantified at the same visits and cell type as stated for total HIV DNA. The real-time PCR assay specific for the 2-LTR junction used primers specified in supplementary table 1 at a concentration of 280 nM and a LNA probe (Mf374) at a concentration of 200 nM. The 2-LTR standard curve was a TOPO cloned 2-LTR DNA insert from an infected HUT78cell line. PCR conditions were per the total assay with the second step increased to 45 cycles. The CV for this assay was 22%.
Integrated HIV DNA Quantification:
Integrated HIV DNA was quantified at weeks 0, 2, 4, 12, 24 and 52 from CD4þT cell DNA using a nested real-time PCR as previously described (Chomont N et al, NM, 2010). 300 nM of Alu 1 and 2 primers and 100 nM of the lambda heel primer L-M667 were used in the first round of 1 cycle of 948C for 7 minutes, 12 cycles of 948C for 30 seconds, 608C for 30 seconds and 728C for 3 minutes and 1 cycle of 728C for 7 minutes on an Eppendorf thermocycler. In the second round 300 nM of primers Lamda T and AA55 M were used with 200 nM of a dual-labeled fluorogenic probe, LTR FL on a BioRad iQ-5 real-time PCR machine. PCR conditions of 1 cycle of 5 minutes at 958C, followed by 45 cycles of 958C
for 15 seconds and 608C for 1 minute. Samples were run quadruplicate in the first round PCR and these were further performed in duplicate in the second round PCR. The CV of this assay was 26%.
Plasma HIV RNA quantitation:
Virus from seven ml of plasma along with the RSV control was pelleted and RNA isolation, cDNA synthesis and PCR were carried out as described (13). Lower limit of detection of the assay is 0.3 copy/ml. When a sample had less than one copy per ml it was depicted as 1 copy/ml and the samples negative in the assay were shown as undetectable.
Flow cytometry:
CD4þand CD8þT cell activation was determined by the expression of cell surface markers (HLA-DR and CD38) for immune activation. Central vs effector memory cells were defined by the expression of the surface marker CD27, with central and transitional memory T cells being CD3þCD4þCD27þ, effector memory cells being CD3þCD4þCD27-.
Supplementary table 1)
Primer Primer Sequence (5’ -3’) Assay
KM Gag Seq F2 TGGTACATCAGGCCATATCACCTAGAACTT Total HIV DNA Assay Gag Sequencing
SK39 TTTGGTCCTTGTCTTATGTCCAGAATGC
SK145 AGTGGGGGGACATCAAGCAGCCATGCAAAT Total HIV DNA Assay
SKCC1B
SKLNA2-3-HIVgag 6-FAM-AT[C]A[A]T[G]AGGAA[G]CT[G]C-BHQ-1
11001 gag F GGTGGGGGGACATCAAGCAGCCATGCAAAT 11001 Patient Specific Total HIV DNA Assay 11001 gag R TACTAGTGGTTCCTGCTATGTCACTTCC
11001 TG gag Probe 6-FAM-AT[T]A[A]T[G]AGGAG[G]CT[G]C-BHQ-1
2-LTR J F GCTAACTAGGGAACCCACTGCTTAAG Episomal 2-LTR HIV DNA Assay HIV R (Brussel A et al.
AIDS 2003) ACTGGTACTAGCTTGTAGCACCATCCA 2-LTR Mf374 Probe 6-FAM-ACA[C]A[C]A[A]G[G][C]T BHQ-1
Alu 1 TCCCAGCTACTGGGGAGGCTGAGG Integrated HIV DNA Assay Round 1
Alu 2 GCCTCCCAAAGTGCTGGGATTACAG
L-M667 ATGCCACGTAAGCGAAACTCTGGCTAACTAGGGAACCCACTG
AA55M GCTAGAGATTTTCCACACTGACTAA Integrated HIV DNA Assay Round 2
Lambda T ATGCCACGTAAGCGAAACT
LTR FL 6-FAM-CACAACAGACGGGCACACACTACTTGA-BHQ-1
