Supporting Information Hatziioannou et al. 10.1073/pnas.0812587106 SI Text Viral Constructs and Viral Stock Generation. To generate the HIV-1 constructs used throughout these studies, the env gene in an HIV-1 NL4 3 proviral plasmid was replaced by the rhesus macaque-adapted KB9 envelope (obtained from pshiv-kb9) using KpnI and BamHI restriction enzymes. To generate the sthiv-1 SV and sthiv-1 2V proviral plasmids, the HIV-1 vif gene in the aforementioned HIV-1 construct was replaced with SIV MAC239 vif and HIV-2 ROD vif, respectively. These constructs were designed so that the 5 end of SIV MAC239 vif or HIV-2 ROD vif was positioned immediately 3 to the HIV-1 pol stop codon. The 5 end of the HIV-1 Vpr coding sequence is positioned immediately 3 to the SIV MAC239 vif and HIV-2 ROD vif stop codons. Additionally, several ATG codons found in the portion of the HIV-1 genome where HIV-1 Vif and Pol would normally overlap, as well as ATG codons normally found within the introduced vif genes, were mutated without changing the amino acid coding potential of HIV-1 Pol or SIV MAC /HIV-2 Vif, respectively. These proviral plasmids were constructed using recombinant PCR approaches. HIV-1, sthiv-1 SV, sthiv-1 2V, SIV MNE023, and SHIV KB9 stocks were generated by transfection using polyethelenimine or Lipofectamine 2000 in 293T cells. In some cases, viral stocks were expanded by brief passage in CEMx174 or HuTCCR5 cells. Viral stocks were titered using GHOST-X4/R5 cells. In Vitro Replication Assays. Spreading replication assays in pigtailed macaque lymphocytes were initiated by inoculating 5 10 5 peripheral blood mononuclear cells, isolated from peripheral blood by Ficoll gradient centrifugation, with a volume of virus stock corresponding to 1 ng of RT. Cells were washed 3 times thereafter, and supernatant samples were harvested every 2 or 3 days for measurement of RT activity using an ELISA-based assay (Cavidi Tech). Animal Inoculation and Monitoring. Pig-tailed macaques (Macaca nemestrina) were housed in accordance with American Association for Accreditation of Laboratory Animal Care standards and all animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committees of the National Cancer Institute or the Tulane National Primate Research Center. All animals were negative for serum antibodies to HIV-2, SIV, simian type-d retrovirus, and simian T-lymphotropic virus type 1. Infections and blood draws were conducted while the animals were sedated. In each infection experiment (Figs. 2A and 5B), infection was initiated by i.v. infusion (saphenous vein) of 1 10 6 i.u. each of sthiv-1 SV and sthiv-1 2V,or2 10 6 i.u of HIV-1 (titers determined using GHOST-X4/R5 cells). Blood was drawn into EDTA- or heparintreated tubes at the time points indicated. Plasma was separated from the blood by centrifugation and was frozen at 80 C in aliquots before analysis for the presence of vrna or antibodies. Cell-Free and Cell-Associated vrna and DNA Measurements. Virions were pelleted from plasma and virion-associated RNA extracted as described previously (1). To isolate cell-associated viral nucleic acids, PBMC and LNMC were sonicated in 3M GuHCl/ proteinase K (1 mg/ml) solution and incubated at 37 C for 1 h. Viral nucleic acids were extracted with a solution of 5.7 M GuSCN, 50 mm TrisCl, ph 7.6, 1 mm EDTA, 0.5% Tween 20, and precipitated with isopropanol with glycogen as carrier and washed with ethanol. Nucleic acids thus obtained were treated with DNase for RNA preparation, or boiled briefly to eliminate RNA before the DNA duplex PCR assay. Plasma viral load or cell-associated viral DNA were quantified by a real time RT-PCR or PCR assay based on amplification of a HIV-1 NL4 3-derived sequence located in the Gag coding region of sthiv-1 SV and sthiv-1 2V. RT was performed with random hexamers and SuperScript II reverse transcriptase (Invitrogen). For the real time PCR amplification, the forward primer was 5 CTA GAA CGA TTC GCA GTT AAT CCT 3, the reverse primer was 5 CTA TCC TTT GAT GCA CAC AAT AGAG3, and the FRET probe was 5 FAM-TCCCAGTATT- TGTCTACAGCCTTCTGATG-BHQ 3. Each PCR mix contained cdna templates, 1 PCR II buffer (ABI), 4.5 mm MgCl2, 0.6 M primers, 0.1 M probe, and 1.25 units AmpliTaq Gold DNA polymerase (ABI). PCR reactions were performed on ABI 7500 Sequence Detection System, (1 cycle of 95 C for 10 min followed by 45 cycles of 95 C for 15 seconds and 60 C for 1 min). Fluorescent signal-based quantification of vrna copy numbers in test samples were determined by ABI 7500 System SDS software, using a standard curve constructed from serial dilutions of an appropriate RNA- or DNA-positive control template. The copy numbers for viral DNA and that of the gene sequence for macaque CCR5 in the same samples, as a reference for diploid genome cell equivalents, were determined in a duplex format quantitative real time PCR assay. The assay was as described above for vrna detection with the omission of RT step, and with the addition of primers and probe (100 nm final concentration) specific for the macaque CCR5 sequence and the use of a cloned genomic fragment of the rhesus macaque CCR5 gene as standard (plasmid pr1-d derived from promoter region of the M. mulatta CCR5 gene). The duplex quantitative PCR assay was run on an MX4000P instrument (Stratagene), and results were reported as nominal sthiv-1 Gag DNA copy numbers per 100,000 cell equivalents. Lymphocyte Subset Monitoring. EDTA-anticoagulated whole blood was incubated with appropriately diluted, directly conjugated monoclonal antibodies against CD3 (Alexa Fluor 488 conjugate), CD4 (APC conjugate), and CD8 (PerCP-Cy5.5 conjugate; all antibodies from BD Biosciences) for 30 min at 4 C, and washed with PBS containing 1% FBS. Red blood cells were lysed with FACS Lyse solution (BD Biosciences) and nucleated cells were washed once more with PBS. Cells were then fixed with 2% paraformaldehyde, and analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, Inc.) or FCS Express (De Novo Software). Absolute lymphocyte subset counts were based on total lymphocyte counts determined by complete blood counts, with differential counts, multiplied by the percentages determined for each lymphocyte subset. Western Blot Analyses. sthiv-1 SV virions were purified by centrifugation through 20% sucrose and resuspended in SDS/PAGE loading buffer. Virion proteins were separated on 4 to 12% acrylamide gels and blotted onto nitrocellulose membranes, which were then cut into strips. The strips were probed with heat-inactivated plasma, diluted 1:200, taken from animals at 0, 2, 4, 8, and 20 weeks after sthiv-1 challenge, or serum from an HIV-1-infected human as a control, followed by an antihuman IgG-peroxidase conjugate. Blots were developed using chemiluminescent detection reagents (Pierce). 1of6
Neutralization Assays. Serially diluted, heat-inactivated plasma was incubated with a fixed inoculum (4 10 3 i.u.) of sthiv-1 SV for 30 min at 37 C. Thereafter, the virus/plasma mixtures were applied to TZM cells for 3 h. The cells were incubated for a further 72 h, lysed, and beta galactosidase activity in cell lysates determined using a chemiluminescent detection assay. Intracellular Cytokine Staining Assays. Cryopreserved peripheral blood mononuclear cells were quick-thawed and placed at 1 10 6 cells/100 l in 96-well round-bottom tissue culture plates. Cells were incubated with pools of 2 g/ml overlapping 15-mer SIVMAC239 accessory or HIV Consensus B accessory, gag, pol, or env gene-encoded peptides (AIDS Research and Reference Reagent Program), in the presence of 1 g/ml anti-cd49d (9F10) and anti-cd28 (CD28.2) costimulatory antibodies (BD Biosciences), for6hat37 C.Media alone was used as a negative control and 5 g/ml Staphylococcus enterotoxin B (Sigma) as a positive control. At the initiation of the culture, 10 g/ml of Brefeldin A (Sigma) was added to all wells. Thereafter, cells were washed with PBS and stained with Violet Fluorescent Reactive Dye (Invitrogen) to exclude dead cells and washed again. Fixation and permeabilization were performed using the BD Biosciences Cytofix/Cytoperm reagents and protocol. Following permeabilization, the cells were resuspended in 100 l of intracellular staining panel containing the BD Biosciences antibodies CD4 PerCP-Cy5.5 (L200), CD8 PE-Cy7 (SK1), CD3 APC-Cy7 (SP34 2), IFN-gamma FITC (B27), IL-2 PE (MQ1 17H12), and TNF-a APC (MAb11). The cells were then washed twice, and 200,000 viable CD3 T-cells were analyzed on an HTS-equipped LSR-II flow cytometer (BD Biosciences). Data analysis was performed using FCS Express. In Vitro Drug Sensitivity Assays. The sensitivity of viruses to inhibition by RT inhibitors was determined by inoculating TZM cells, in duplicate, with HIV-1, SIV MNE027, SHIV KB9, or sthiv-1 in the presence of 10 g/ml of DEAE-dextran, with or without RT inhibitors. Cells were incubated for2hat37 C,washed with sterile PBS, received new medium with or without added inhibitor, and were harvested 48 h later. Cells were lysed with Glo lysis buffer (Promega) and assayed for luminescence with the Luciferase Assay System (Promega) with a LMaxII luminometer (Molecular Devices). Results were expressed as the percentage of the luciferase activity obtained for each virus in the absence of inhibitor. Virus-inhibition assays using protease inhibitors were done by inoculating CEMx174 cells with HIV-1, SHIV KB9, SIV MNE027, or sthiv-1. When syncytia were observed, the cells were washed and cultured overnight in the presence of increasing concentrations of inhibitor. Thereafter, culture supernatants were harvested and used to inoculate TZM cells. Forty-eight hours later, luciferase activity in cell lysates was determined, as described above. 1. Cline AN, Bess JW, Piatak M, Jr., Lifson JD (2005) Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS J Med Primatol 34:303 312. 2of6
Fig. S1. CD3 CD4 counts in 2 of the 4 sthiv-1 infected macaques. 3of6
Fig. S2. Examples of cellular immune responses evaluated by intracellular cytokine staining. CD3 /CD4 and CD3 /CD8 cells from animal PT6356, obtained at the time of infection (week 0) and 4 weeks after infection were analyzed for the production of IFN-gamma and IL-2, following stimulation with peptide pools corresponding to Gag, Pol and accessory genes. No Gag-specific CD8 responses were detected. 4of6
Table S1. IC 50 values a for antiretroviral drugs HIV-1 sthiv-1 SV sthiv-1 2V SHIV KB9 SIV mne FTC b 0.5 0.2 0.6 0 0.5 0 0.5 0.2 1 0 Tenofovir b 4 2 3 0.7 4 0.7 2 0.8 1 0.9 Efavirenz b 3 0.7 2 0.7 2 0.7 150 150 Amprenavir b 0.3 0.5 0.3 ND 5 Atazanavir b 5 11 8 ND 100 a M, FTC, tenofovir, and amprenavir; nm, efavirenz and atazanavir. b Average of 2 independent experiments standard deviation. ND, not done. 5of6
Table S2. PCR assays for cell-associated viral DNA a and RNA a in PrEP-treated macaques and a contemporaneous control PT8242 (PrEP) PT14232 (PrEP) PT8249 (No PrEP) DNA RNA DNA RNA DNA RNA Weeks after challenge PBMC LN PBMC LN PBMC LN PBMC LN PBMC LN PBMC LN 2 30 30 30 30 30 30 30 30 30 30 30 30 1 30 30 30 30 30 30 30 30 30 30 30 310 4 30 30 30 30 30 30 30 30 40 140 30 200 a Values indicate number of copies of viral DNA or RNA per 100,000 cells. 6of6