www.sciencemag.org/content/344/6190/1401/suppl/DC1

Supplementary Materials for

HIV-1–induced AIDS in monkeys

Theodora Hatziioannou,* Gregory Q. Del Prete, Brandon F. Keele, Jacob D. Estes,

Matthew W. McNatt, Julia Bitzegeio, Alice Raymond, Anthony Rodriguez, Fabian

Schmidt, C. Mac Trubey, Jeremy Smedley, Michael Piatak Jr., Vineet N. KewalRamani,*

Jeffrey D. Lifson,* Paul D. Bieniasz*

*Corresponding author. E-mail: thatziio@adarc.org (T.H.); vineet.kewalramani@nih.gov (V.N.K.);

lifsonj@mail.nih.gov (J.D.L.); pbienias@adarc.org (P.D.B.)

Published 20 June 2014, Science 344, 1401 (2014)

DOI: 10.1126/science.1250761

This PDF file includes:

Materials and Methods

Figs. S1 to S8

Full Reference List

Materials and Methods

HIV-1 strains.

A proviral plasmid clone of HIV-1 encoding SIVmac Vif and the macaque adapted

envelope from SHIV/KB9 has previously been described(11). Derivatives of this proviral

plasmid were constructed by replacing Env-encoding sequences with the corresponding

sequences from previously described NL4-3-derived proviral plasmids(26). In all these

chimeric Env proteins, the signal peptide is derived from NL4-3 Env whereas the

remaining gp120 encoding sequences were derived from YU2, BaL, AD8 or

KB9(V3ADA) strains. At the C-terminal end of the introduced R5-tropic Env sequences,

the junction between YU2, BaL or KB9(V3ADA) and NL4-3 Env sequences is at amino

acid 753 (within the Env cytoplasmic tail) while the junction between AD8 and NL4-3 is

at amino acid 590 (in the gp41 ectodomain). The KB9(V3ADA) Env sequence was

constructed using overlapping PCR primers to replace 35 amino acids of the V3 loop in

the KB9 envelope by the corresponding sequence from ADA, which is identical to the

HIV-1 subtype B consensus. We confirmed that all of the aforementioned constructs

generated viruses that replicated in vitro in MT2 cell lines engineered to express CCR5.

To generate HIV-1KB9 Vpu 15/21, Vpu-coding sequences were amplified from a selected

amplicon obtained from the plasma of P3-A and used to replace the corresponding Vpu

sequences in HIVKB9 using overlapping PCR and the EcoRI-KpnI sites in HIV-1NL4-3. A

Vpu-defective HIV-1NL4-3 construct has been previously described(21).

HIV-1 adaptation to pigtailed macaques

Pigtailed macaques (Macacca nemestrina) were housed and cared for in accordance with

American Association for Accreditation of Laboratory Animal Care (AAALAC)

standards in an AAALAC-accredited facility and all animal procedures were performed

according to a protocol approved by the Institutional Animal Care and Use Committee of

the National Cancer Institute. At the start of the study, all animals were free of

cercopithicine herpesvirus 1, simian immunodeficiency virus (SIV), simian type-D

retrovirus, and simian T-lymphotropic virus type 1. Plasma for viral RNA (vRNA)

quantification, sequencing analysis and Western blots, and peripheral blood mononuclear

cells (PBMCs) for flow cytometry assays were isolated from whole blood collected in

EDTA-anticoagulated Vacutainer tubes (BD) 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. PBMCs were isolated from whole

EDTA blood by Ficoll-Paque Plus (GE Healthcare) gradient centrifugation.

To initiate adaptation of HIV-1 to macaques, P1-A and P1-B were intravenously (IV)

inoculated (saphenous vein) with a cocktail of cell culture supernatants from transfected

293T cells that contained 5x105 i.u. of each HIV-1 variant. During subsequent serial

passages, some of the animals, as noted below, received subcutaneous administration

(s.c.) of either a chimeric ‘humanized’ anti-CD8 monoclonal antibody (MAb), cM-T807,

or a chimeric ‘rhesusized’ anti-CD8 MAb, M-T807R1 (both obtained through the NCRR

non-human primate reagent program, from Keith Reimann, Harvard/Beth Israel

Deaconess Medical Center), at the dosages indicated. For serial passage of blood from

infected to naïve macaques, whole blood from donor animals was drawn into acid citrate

dextrose (ACD) Vacutainer tubes immediately prior to i.v. infusion into recipients. At

weeks 32 and 33 after infection, P1-A received cM-T807 at 25 mg/kg of body weight.

Thereafter, 10ml of blood withdrawn from P1-A at week 34 after infection was used to

inoculate P2-A. P2-A received s.c. injection of cM-T807 at 25 mg/kg of body weight at

the time of infection and at one week postinfection (pi). Blood (10ml) obtained from P2-

A at week 23 pi, immediately prior to administration of M-T807R1 at 50 mg/kg, was

used to inoculate animal P3-A and blood (10ml) obtained from P2-A at week 24 pi, one

week after M-T807R1 administration, was used to inoculate P3-B. Both P3-A and P3-B

received M-T807R1 at 25 mg/kg of body weight at the time of inoculation and one week

later. Thereafter, P3-A and P3-B received M-T807R1 at 25 mg/kg of body weight at

weeks 12 and 13 or 11 and 12, for P3-A and P3-B, respectively, and at 50 mg/kg of body

weight at week 27 or 26, for P3-A and P3-B, respectively. Passage 4 was initiated using

blood obtained from P3-A and P3-B at weeks 47 and 46 after infection, respectively, one

week after administration of M-T807R1 at 50 mg/kg. P4-A was inoculated with 10ml

blood obtained from P3-A, P4-B was inoculated with blood from both P3-A and P3-B

(5ml each) and P4-C was inoculated with 10ml blood from P3-B. M-T807R1 was

administered at 25 mg/kg to P4-A, P4-B and P4-C at the time of inoculation and at one

week post inoculation. For passage 5, blood obtained from animal P4-C at week 28 after

infection (necropsy) was used to inoculate four animals (10ml blood per animal). P5-A

and P5-B were not subjected to CD8+ T-cell depletion, while P5-C and P5-D were

injected with M-T807R1 at the time of inoculation and one week thereafter. M-T807R1

was subsequently administered to P5-A and P5-B at weeks 23 and 24 pi. Blood (10ml)

obtained from P5-C at week 39 after infection was used to inoculate P6-A, that was not

subjected to CD8+ T-cell depletion, and P6-B that was injected with M-T807R1 at the

time of inoculation and one week thereafter.

Viral load measurements

Virions were pelleted from plasma and virion-associated RNA extracted as described

previously(11). Plasma viral load was quantified using real time qRT-PCR, based on

amplification of an HIV-1NL4-3-derived sequence located in the Gag coding region.

Reverse transcription 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 AGA G 3’ and the FRET probe was 5’ FAM

TCCCAGTATTTGTCTACAGCCTTCTGATG-BHQ 3’. Each PCR mix contained

cDNA templates, 1x PCR II buffer (ABI), 4.5 mM MgCl2, 0.6 μM primers, 0.1 μM

probe, and 1.25 units AmpliTaq Gold DNA polymerase (ABI). PCR reactions were

performed on an 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 control template.

Flow cytometry

Antibodies and reagents were obtained from BD Biosciences, unless indicated otherwise

and data analysis was performed using FCS Express (De Novo Software). Sample

preparation absolute cell counting and lymphocyte immunophenotyping methods were

performed as previously described(11, 27, 28) Briefly, absolute cell counts were

performed on EDTA-anti-coagulated whole blood using the following surface antigen

staining panel: CD45 FITC (DO58-1283), CD3 PE (SP34-2), CD4 APC (L200), CD14

APC-Cy7 (M5E2; BioLegend), CD8α PE-Cy7 (SK1), and CD20 Pacific Blue (2H7;

BioLegend). FACS Lysing solution (BD Biosciences) was then added and approximately

50,000 CD45+CD3+ cells were acquired for each sample, using a BD FACSVerse flow

cytometer equipped with a volumetric flow sensor. Lymphocyte immunophenotyping

was performed on freshly isolated mononuclear cells using the following antibodies: CD4

Pacific Blue (OKT4; BioLegend), CCR5 PE (3A9), CD28 ECD (CD28.2; Beckman

Coulter), CD95 PE-Cy5 (DX2), CD8 PE-Cy7 (SK1), CD38 APC (OK10; NIH

Nonhuman Primate Resource), CD3 APC-Cy7 (SP34-2), and Ki67 FITC (B56). Surface

and intracellular staining was performed using BD Cytofix/Cytoperm reagents and

protocol. Approximately 200,000 CD3+ T-cells were acquired for each sample using a

BD LSR-II flow cytometer.

Western blot analyses

HIV-1 (AD8) virions, generated by transfection of 293T cells with a proviral plasmid

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 from infected macaques, diluted 1:200, or serum from an

HIV-1-infected human as a control, followed by an anti-human IgG-peroxidase

conjugate. Blots were developed using chemiluminescent detection reagents (Pierce).

Single genome amplification/sequencing and phylogenetic analysis

Sequences were obtained from plasma viral RNA and PCR amplified using the single

genome amplification technique as previously described(18). The entire 3’ half of the

viral genome (including the entire vif, vpr, vpu, tat, rev env and nef genes) was amplified

from cDNA generated by reverse transcription of RNA using SuperScript III reverse

transcriptase according to the manufacturer’s recommendations (Invitrogen). In brief, a

cDNA reaction of 1× RT buffer, 0.5 mM of each deoxynucleoside triphosphate, 5 mM

dithiothreitol, 2 U/ml RNaseOUT (RNase inhibitor), 10 U/ml of SuperScript III reverse

transcriptase, and 0.25 mM antisense primer HIVR3B3.R1 5’-

ACTACTTGAAGCACTCAAGGCAAGCTTTATTG-3’ was incubated at 50°C for 60

min, 55°C for 60 min and then heat-inactivated at 70°C for 15 min followed by treatment

with 1 U of RNase H at 37°C for 20 min. Thereafter, cDNA was amplified via limiting

dilution PCR where only one amplifiable molecule was present in each reaction using 1×

PCR buffer, 2mM MgCl2, 0.2mM of each deoxynucleoside triphosphate, 0.2μM of each

primer, and 0.025 U/μl Platinum Taq polymerase (Invitrogen) in a 20-μl reaction. First

round PCR was performed with sense primer HIVBK3F1 5’-

ACAGCAGTACAAATGGCAGTATT-3’ and antisense primer HIVR3B3.R1 under the

following conditions: 1 cycle of 94°C for 2 min, 35 cycles at 94°C for 15 sec, 55°C for

30 sec, and 72°C for 4 min, followed by a final extension of 72°C for 10 min. Next, 1μl

from the first-round PCR product was added to a second-round PCR reaction that

included the sense primer HIVBK3F2 5’- TGGAAAGGTGAAGGGGCAGT-

AGTAATAC-3’ and antisense primer HIVR3B6.R2 5’-

TGAAGCACTCAAGGCAAGCTTTA-TTGAGGC-3’ performed under the same

conditions used for first-round PCR, but with a total of 45 cycles. For 5’ genome

sequences including Gag and Pol, the following primers were used: HIVBK5R1 5’-

CTTGCCACACAATCATCACCTGCCATCTG-3’, HIVBK5R2 5’-CAATCA-

TCACCTGCCATCTGTTTTCCATA-3’, HIVU5B1F1 5’-

CCTTGAGTGCTTCAAGTAGTGT-GTGCCCGTCTGT-3’, and HIVU5B4F2 5’-

GTAGTGTGTGCCCGTCTGTTGTGTGACTC-3’. Correct sized amplicons were

identified by agarose gel electrophoresis and directly sequenced with second round PCR

primers and HIV-1 specific primers using BigDye Terminator technology. Sequences

were aligned using ClustalW and hand edited using MacClade 4.08 to improve alignment

quality. Trees were constructed using the neighbor-joining method.

Immunohistochemistry and In situ hybridization analysis

Immunohistochemistry (IHC) was performed using a biotin-free polymer approach

(Golden Bridge International, Inc.) and 5μm tissue sections, mounted on glass slides as

previously described(27). For CD4+ T cell IHC, heat induced epitope retrieval (HIER)

was performed by heating sections in 0.01% citraconic anhydride containing 0.05%

Tween-20 in a pressure cooker (Biocare Medical) set at 122°C for 30 s. Slides were

incubated with blocking buffer (TBS with 0.05% Tween-20 and 0.25% casein) for 10min

and then incubated with mouse anti-CD68 (1:400; clone KP1, Dako), mouse anti-CD163

(1:400; clone 10D6; Novocastra/Leica) and rabbit monoclonal anti-CD4 (1:200; clone

EPR6855; Abcam, Inc.) diluted in blocking buffer overnight at 4oC. Slides were washed

in 1X TBS with 0.05% Tween-20 and endogenous peroxidases blocked using 1.5% (v/v)

H2O2 in TBS (pH 7.4) for 10min. Slides were incubated Mouse Polink-1 AP followed by

Rabbit Polink-1 horseradish peroxidase (HRP, Golden Bridge International, Inc.) for 30

min each at room temperature. Tissue sections were first incubated with 3,3′-

diaminobenzidine (Impact™ DAB, Vector Laboratories) to reveal CD4, washed and

developed with Warp Red (Biocare Medical, Inc.) to mask the low levels of CD4

expressed on myeloid cells, allowing for specific identification of CD4+ T cells.

Identification of B-cell lymphomas was performed by performing double IHC for CD3

(Warp Red) and CD20 (DAB) in a manner analogous to the CD4+ T cell IHC described

above, but utilizing Mouse Polink-1 HRP followed by Rabbit Polink-1 alakaline

phosphatase (ALP). Slides were washed in ddH2O, counterstained with hematoxylin,

mounted in Permount (Fisher Scientific), and scanned at high magnification (x200) using

the ScanScope CS System (Aperio Technologies) yielding high-resolution images from

the entire tissue section. Representative regions of interest (500 m2) were identified and

high-resolution images extracted from the whole-tissue scans. In situ hybridization

(chromogenic and fluorescent) analysis was performed as previously described using

HIV-1 clade B lineage specific riboprobes(29, 30). Phenotypic analysis of the cell types

productively infected with HIV-1 was performed by manually counting the HIV-1

vRNA+ cells from confocal images taken on an Olympus FV10i confocal microscope

using a 60X oil-immersion objective (NA 1.35) as previously described(30). Lung tissue

was stained using the Grocott's methenamine silver stain to identify fungal organisms, in

particular Pneumocystis which causes Pneumocystis Pneumonia (PCP), a classical AIDS-

defining illness. Antibodies used for IHC in this study were rabbit monoclonal anti-

human CD3 (clone SP7; Thermo Scientific), rabbit monoclonal anti-human CD4 (clone

EPR6855; Abcam), mouse anti-human CD20 (clone L26; Dako), mouse anti-human

CD68 (clone KP1; Dako), mouse anti-human CD163 (clone NCL-L-CD163;

Novocastra/Leica), mouse anti-human collagen I (clone COL-1; Sigma-Aldrich); mouse

anti-human collagen III (clone FH-7A; Sigma-Aldrich); and rabbit monoclonal anti-

human Ki67 (clone SP6; Lab Vision/Thermo Fisher Scientific).

Analysis of adapted envelope function and inhibitor sensitivity

Env coding sequences, derived from P5-B plasma 3’ viral clones were inserted into

pcDNA3.1/V5-His-TOPO (Invitrogen). Control plasmids expressing the same cassette

amplified from the HIV-1AD8 or HIV-1NL4-3 proviral construct were also generated. These

Env-expressing plasmids were co-transfected with a proviral HIV-1ΔEnv/GFP plasmid in

293T cells. Clarified and filtered supernatants, harvested 48h post-transfection, were used

to infect MT2-R5 cells in absence or presence of Maraviroc (8nM) or AMD3100 (1μM).

Numbers of GFP positive cells obtained in the absence of drugs were set to100% for each

virus and percentage of infection in the presence of drugs is presented.

Analysis of Vpu function

To assess the ability of Vpu to antagonize tetherin, HIV-1 proviral plasmids (HIV-1NL4-3,

HIV-1NL4-3 delVpu or HIV-1KB9Vpu 15/21) were cotransfected in 293T cells with

increasing amounts of plasmids expressing human tetherin, or three naturally occurring

variants of pigtailed macaque tetherin(21). At 48h after transfection, the infectious virion

yield was determined by applying clarified and filtered cell culture supernatants to TZM-

bl cells, and measuring β-galactosidase activity in cell lysates, 48h later.

Fig. S1.

Schematic representation of the HIV-1 constructs used in this study. White and black

regions indicate HIV-1 and SIVMAC239-derived sequences respectively. Env proteins were

derived from prototype R5-tropic HIV-1 strains (AD8, YU2, BaL) or the macaque-

adapted KB9 envelope with the V3 loop substituted with that from ADA (KB9V3ADA).

See methods for details of construction.

Fig. S2

CD8+ T-cell counts in the blood of infected macaques. Absolute numbers of CD8+CD3+

cells were determined by flow cytometry. Color-coded arrowheads indicate the times at

which the first of two doses (1 week apart) of a CD8 antibody (either cM-T807 or M-

T807R1) was administered.

Fig. S3

CD8+ T-cell subset frequencies in GALT of HIV-1 infected macaques. Cell suspensions

derived from GALT specimens obtained at the indicated times after infection were

analyzed by flow cytometry using antibodies against CD3, CD4, CD8, CD28 and CD95.

The percentage of CD3+ cells that also expressed CD8 and central memory (CD95+,

CD28+, effector memory (CD95+, CD28-), or naïve markers (CD95-, CD28+) is plotted.

Fig. S4

(A) Immune activation in GALT CD4+ cells. Cell suspensions derived from GALT

specimens obtained at the indicated times after infection were analyzed by flow

cytometery using antibodies against CD3, CD4 and Ki67. The percentage of CD3+CD4+

cells that also expressed Ki67 is plotted. (B) Immmunohistochemical stain for Ki67

antigen (brown) in lymph node sections taken from P4 animals pre infection or 18

weeeks after after infection. (C) Immmunohistochemical stain for collagen I, (brown) in

lymph node sections from the same specimens as in (B). (D) Immmunohistochemical

stain for collagen III (brown) in lymph node sections from the same specimens as in (B).

Fig. S5

Characterization of AIDS-defining B-cell lymphoma found in P4-C. (A) Hematoxylin

and eosin stained representative tumor sections from retro-orbital, spinal and renal

masses. Scale bars = 5mm (top row) 1mm (second row) 400 μm (third row) and 100 μm

(fourth row). (B) Immunohistochemical staining of CD20+ B-cells (brown) and CD3+ T-

cells (red) at the three tumor sites. Scale bars = 100μm. (C) Negative control stains

(brown) for CD3+ T-cells (upper panels) and CD20+ B-cells lower panels from

unaffected tissues (Kidney) taken from clinically well macaques. Scale bars = 200μm.

Fig. S6

Detection of Pneumocystis organisms in lungs of macaques subjected to Grocott's

methenamine silver stain. (A) Control sections from clinically well macaques P2-A and

P3-B (compare with Fig. 2E). (B) Detection of Pnuemocystis organisms in lung sections

from P5-C (black spots, right panel). A control Pneumocystis-negative lung specimen

(from P5-A, left panel) is also shown for comparison. Scale bars = 50μm.

Fig. S7

Cell types infected by HIV-1 in macaques. (A) In situ hybridization analysis to detect

infected HIV-1 RNA-positive cells (brown) in lymph nodes and GALT of an infected

macaque (P4-C), Scale bars = 100μm. (B) Example of a lymph node section, from P4-C,

subjected to fluorescent in-situ hybridization (FISH) to detect HIV-1 infected cells (red)

and immunofluorescence to detect macrophages (CD68+/CD163+; green) and T-cells

(CD3+, blue). Examples of infected T-cells and macrophages are indicated by black and

white arrows respectively. Scale bars = 50μm. (C) Phenotypic analysis of the cell types

(CD3+ T-cells or CD68+/CD163+ macrophages) productively infected by HIV-1 in

lymph node and GALT sections from infected macaques, at the indicated times after

infection. The number of infected cells analyzed is given for each animal and time point.

Fig. S8

Antibody responses to HIV-1 in pigtail macaques. Western blot analyses were performed

using purified HIV-1 virions (encoding AD8 Env) as the antigen and using plasma

samples recovered from the HIV-1 infected macaques at the indicated number of weeks

after infection. Serum from an HIV-1-infected human long-term non-progressor (+ve)

was used as a positive control.

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