Peridinin

Cell culture and reagents: SUPT1/CCR5 CL.30 cells (provided by J. Hoxie, University of Pennsylvania) and 174×CEM cells (AIDS Research and Reagent Program, courtesy of Peter Cresswell) were maintained in RPMI 1640 (Cellgro; Fisher Scientific, Springfield, N.J.) with 10% (v/v) heat-inactivated fetal calf serum (FCS, Cellgro). The TZM.bl cell line (AIDS Research and Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc) was maintained in DMEM (Cellgro) with 10% (v/v) heat-inactivated FCS. Peripheral blood mononuclear cells (PBMCs) were isolated from HIV seronegative leukocyte-enriched preparations purchased from the New York Blood Center using Ficoll-Hypaque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden). Monocytes were isolated using CD14 magnetic cell sorting (Miltenyi Biotec, Auburn, CA), with washing and elution in cold 1× PBS supplemented with 1% AB human serum (Cellgro) and 1 mM EDTA (Sigma). Monocyte purity was verified in each experiment by CD14 (MP9) and CD3 (Leu-4) staining (both Becton Dickinson, San Jose, CA), with cut-off purities of 2% CD3 T cells. Monocytes were subsequently cultured in RPMI 1640 (Cellgro) containing 10 mM HEPES (GIBCO-BRL, Life Technologies, Grand Island, NY), 2 mM L-glutamine (GIBCO-BRL), 50 μM 2-mercaptoethanol (Sigma, St. Louis, MO), penicillin (100 U/ml)-streptomycin (100 μg/ml) (GIBCO-BRL), and 1% heparinized human plasma (Innovative Research, Southfield, MI) supplemented with 100 U/ml recombinant human interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN) and 1000 U/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Biosource/Invitrogen, Carlsbad, CA). To generate mature DCs, day 5-cultured immature DCs were exposed to a maturation cocktail of IL-1β (10 ng/ml), IL-6 (1,000 U/ml), TNF-α (10 ng/ml) (all from R&D Systems, Minneapolis, MI) and PGE₂ (1 μg/ml) (Sigma) for 48 hours. The phenotype of immature and mature DCs was routinely monitored by two-color flow cytometry using FITC-conjugated mouse Ab against HLA-DR (Becton Dickinson) combined with the following panel of phycoerythrin (PE)-conjugated mouse anti-human monoclonal Abs (MAbs): anti-CD25, -CD80, -CD86 (all Becton Dickinson), and -CD83 (PN IM2218; Immunotech, Marseille, France).
Microbicide preparations: Carraguard (Lot numbers 032805, 102505, 032906-A, 011005-B, and 010908) was prepared as a 3% (w/v) stock as described [34] (link). PC-817 (Lot numbers 032805, 102705, 040306-B, 011005, and 032707-A) was prepared adding a DMSO (Sigma) or ethanol solution of MIV-150 (Medivir AB, Sweden) to Carraguard, to a final concentration of 500 μM. 2.5% (25 mg/ml) methylcellulose (MC; Lot numbers 032805, 110205, 033006-A, 011005-A, 032807, and 011008) (Fisher) was used as a placebo vehicle control gel for the in vivo studies. To test the in vivo activity of MIV-150 alone, MIV-150 was mixed with 25 mg/ml MC (Lot numbers 040306-A, 032707B, and 011908). All gels were stored at room temperature. In vitro assays with MIV-150 were set up using 10 mM MIV-150 stocks dissolved in DMSO. 3% Carraguard stock solutions were diluted initially 1∶10 (v/v) with 1× PBS using a positive displacement pipette (Eppendorf, Hamburg, Germany).
Virus stocks and titering: HIV_MN and HIV_Bal stocks were sucrose gradient purified lots #P3764 and #P3953 (courtesy of the AIDS and Cancer Virus Program, SAIC-Frederick, Inc., National Cancer Institute, Frederick, MD). The RT-SHIV construct is a hybrid of SIVmac239 bearing the reverse transcriptase gene derived from HIV HXB2 [38] (link), [39] (link). RT-SHIV stocks for in vivo inoculations were grown in PHA activated human PBMCs (kindly provided by Disa Böttiger, Medivir AB, Sweden). RT-SHIV stocks were titered using the 174×CEM cell line and TCID₅₀ was calculated according to the Reed and Muench formula.
For in vitro assays, a purified and high titer stock of RT-SHIV was generated as follows: 4 liters of viral supernatant were produced in the 174×CEM cell line and harvested over a period of 28 days. Viral supernatant was pre-cleared of cellular debris, by centrifugation at 1800×g for 30 min at 4°C using a benchtop centrifuge (Eppendorf). Virus was then concentrated 100 fold, using a Labscale tangential filter flow apparatus connected in parallel with two Pellicon XL 50 Cassettes with 1000 kDa molecular weight cut-off (Millipore, Billerica, MA). For 37 ml of virus filter concentrate, virus pellets were generated by ultracentrifugation in a SW28 rotor (Beckman-Coulter, Fullerton, CA) at 100,000 g through a 1 ml 20% glycerol cushion and then virus was resuspended in 400 μl of PBS and layered onto a 9 step 24% to 56% sucrose gradient. Virus was subsequently ultracentrifuged in a SW55Ti rotor (Beckman-Coulter) at 100,000×g for 3 hours with acceleration and deceleration set at 5 and 9 respectively. For sucrose gradients, peak viral fractions were harvested by analyzing A₂₈₀ using a spectrophotometer and were later confirmed to correspond to peak infectivity using the TZM.bl cell line [15] (link). Harvested fractions were diluted 1 in 5 ml in 1× PBS and subsequently pelleted at 100,000×g for 90 min in a SW55 rotor (Beckman-Coulter). The pellets were resuspended overnight in 1 ml of PBS and stored at −80°C. The titer (2.49×10⁸ TCID₅₀/ml) was determined using 174×CEM cells as described above.
HIV/SIV infections and mature DC transfer assays: TZM.bl cells (plated at 5×10³ cells/well in 96 well flat-bottomed plates 16 hours earlier) or PBMCs activated for 48 hours with 5 μg/ml PHA (Sigma) (10⁶ cells/ml in 200 μl in 96 well round-bottomed plates) were treated with compounds for 30 min at 37°C and then challenged with 300 TCID₅₀ of HIV_Bal or HIV_MN or 600 TCID₅₀ of RT-SHIV. Activated PBMCs were recultured with complete media supplemented with 10 U/ml of IL2(Roche). For immature and mature DCs, 1.5×10⁵ cells (in 150 μl) were pretreated with compounds and pulsed with either 3000 TCID₅₀ of HIV_Bal, 4500 TCID₅₀ of RT-SHIV or 3000 TCID₅₀ of VSVg pseudotyped, delta HIV envelope NL43 in 96 well V-bottomed plates (Corning, NY). After 2 hours at 37°C, mature DCs were washed 4 times in media and then 10³ DCs were added to 5×10³ TZM.bl indicator cells or 5×10³ DCs were also added to equal numbers of either SUPT1/CCR5 CL.30 cells (for HIV_Bal) or 174×CEM cells (for RT-SHIV). Detection of virus transfer to TZM.bl cells was by X-gal staining as described [15] (link), [40] (link). Detection of transfer to SUPT1/CCR5 CL.30 cells was by Q-PCR for HIV gag DNA and RT-SHIV transfer to 174×CEM cells was by Q-PCR for SIV gag DNA, as a function of cell numbers by using Q-PCR for albumin DNA [41] (link), [42] (link). Virus-pulsed immature DCs were washed before being cultured in 96 well round-bottomed plates at 10⁶ cells/ml in 200 μl of IL-4/GM-CSF media. Infection of immature DCs was monitored using intracellular stain for HIV gag p24 [43] (link). The percent inhibition of infection was calculated using the following equation:
Microbicide application and in vivo challenge: Adult female Chinese rhesus macaques (Macaca mulatta) were housed at the Tulane National Primate Research Center (TNPRC; Covington, LA). All studies were approved by the Animal Care and Use Committee of the TNPRC. Animal care procedures were in compliance with the regulations detailed in the Animal Welfare Act [44] and in the “Guide for the Care and Use of Laboratory Animals” [45] . All naïve animals tested negative for simian type D retroviruses, simian T cell leukemia virus-1, and SIV prior to use. Prior to virus challenge, animals received a single 30 mg i.m. injection of Depo-Provera. 35 days later, the macaques were sedated and 3 ml of compound were introduced atraumatically into the vaginal vault using a pliable French catheter. 1 ml of virus was applied 30 min later. At appropriate time points, pre and post viral challenge, animals were anesthetized with ketamine-HCl (10 mg/kg) prior to EDTA blood samples being taken (no more than 10 ml/kg/month/animal).
Anti-CD8 depletion: Monkeys were treated with the mouse-human chimeric anti-CD8 mAb cM-T807 (NIH Nonhuman Primate Reagent Resource-Beth Israel Deaconess Medical Center, Boston, MA), receiving 10 mg/ml s.c. at day 0, followed by 5 mg/kg i.v. on days 3, 7, and 10 [46] (link). To verify CD8 cell depletion, whole blood was stained according to the manufacturer's guidelines for phycoerythrin (PE)-conjugated anti-CD8 (clone DK25; BD Pharmingen), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (clone L200; Dako), peridinin-chlorophyll-Cychrome (PerCP-Cy5.5)-conjugated anti-CD3 (clone SP34; BD Pharmingen).
Plasma viral load: Plasma was collected from whole EDTA blood after bench top centrifugation (Eppendorf) at 800×g for 10 min. Contaminating platelets were removed by a second centrifugation at 800×g for 10 min. Plasma was then stored in 1 ml aliquots at −80°C until plasma viral load RNA detection. Measurement of plasma viral loads by quantitative RT-PCR was performed as previously described [47] (link), [48] (link). We defined animals that were “infected” as those which recorded greater than 1000 RNA copies/ml in ≥2 samples post infection. Animals defined as “uninfected” had undectectable viral RNA for the duration of the viral challenge study (20 weeks) or <1000 RNA copies per ml at <2 time points post challenge.
ELISPOT assay: ELISPOT assays were performed as previously described [47] (link), [49] using 300 ng p27/ml of AT-2 inactivated SIVmneE11S [50] (link) (Lot# p3926, courtesy of the AIDS and Cancer Virus Program, SAIC-Frederick) as the SIV antigen (vs the no virus microvesicle controls). SIV-specific responses were determined by subtracting the responses detected in control cultures from those induced by AT-2 SIV. In each experiment, PBMCs were also cultured with 5 μg/ml Concanavalin A (Sigma) to control for PBMC functionality and assay integrity. Spots were counted using an AID ELISPOT reader (Cell Technology, Columbia, MD) with once optimized settings through all experiments and the mean (±SEM) numbers of spot forming cells (SFCs) from triplicate or duplicate cultures per animal were enumerated.
SIV specific antibody response: Plasma samples obtained were monitored for the presence of SIV envelope Abs by using an established ELISA protocol [51] (link).
Whole blood CD8/CD4 T-cell counts: Absolute CD4 and CD8 cell counts were monitored by TruCount (BDBiosciences, Palo Alto, CA) staining of whole blood at the indicated time points.
Statistical analyses used in this study: Unless otherwise stated, data was tested for normal distribution using Origin software (Shapiro-Wilk test) (Originlab corporation, Northhampton, MA). For statistical comparisons, 2-tailed and paired t tests were used for the in vitro analyses. Fisher's Exact was calculated for in vivo analyses [52] , with the aid of software published online at http://www.langsrud.com/fisher.htm. Standard p values <0.05 were taken as statistically significant.

Collection and staining of PB samples. PB was drawn (21 G needles) in two sodium citrate tubes (Becton Dickinson Biosciences-BD, San Jose, CA, USA, Ref 454387) and processed within 4 h from venepuncture. The first harvested tube was discarded to minimize venepuncture-induced vascular damage effects [55 (link)]. To obtain a method for a rigorous EVs definition, different known EVs tracers were tested (Supplementary Table S3). Among them, LCD resulted in the most promising marker, giving the best separation of the positive population with respect to the related internal negative one (Supplementary Figure S7). The best combination of markers for EVs analysis was obtained after testing different reagent combinations (Supplementary Table S4). In order to stain PB samples, a reagent mix was prepared by adding to 195 μL of PBS 1×, 0.5 μL of Fluorescein isothiocyanate (FITC)-conjugated phalloidin and LCD, and all reagents, as detailed in Supplementary Table S5; then 5 μL of whole blood were added to the mix. The lipophilic cationic dye is a commercial compound, that we have validated and patented for its off label use to stain EVs for further flow cytometry analysis. The chemical structure of this molecule is not public. BD Biosciences produces the custom LCD kit on the basis of customer requests. Given that LCD kit is a custom product, it is not reported on standard catalogues, but related reference numbers are 626266 (antibodies, listed in Supplementary Table S5) and 626267 (LCD and FITC-conjugated phalloidin, Supplementary Table S5). To avoid immune complex formation and the unspecific background linked to the antibody aggregation, each reagent stock solution was centrifuged before its use (21,000× g, 12 min). After 45 min of staining (RT, in the dark, or at 37 °C when Annexin V was not present in the reagent mix), 500 μL of PBS 1× were added to each tube and 1 × 10⁶ events/sample were acquired by flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA). In a subset of samples, Peridinin Chlorophyll Protein-Cyanin (PerCP-Cy) 5.5-conjugated Annexin V was also added (0.25 µL, BD Biosciences, Cat: 561431), and, in this case, Binding Buffer 1X (BD Biosciences) was used instead of PBS 1X. The dilution of the sample was optimized, and, at the used dilution (1:143), no swarm effects occur (Supplementary Figure S5A) [56 (link)].
All requirements imposed for polychromatic flow cytometry EVs analysis were taken into account [18 (link),40 (link),41 (link),48 (link)]. In detail, MISEV guidelines for analytical variables, as well as MIFlowCyt and MIFlowCyt-EV suggestions for general variables and experimental design related to FC EVs experiments were taken into account.

Antibodies employed in this study were from Thermo Fisher Scientific (Waltham, MA). Those for flow cytometry were conjugated to phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Alexa700, PerCP-Cyanine5.5 (PerCp-Cy5) or fluorescein isothiocyanate (FITC): CD29-PerCP, CD73-PE, CD90-Alexa700, CD105-PE, CD166-PE, CD31-PE, CD34-PerCP-Cy5 and CD45-FITC. Antibodies for Cx43 and alpha-tubulin were rabbit polyclonal and monoclonal antibodies, respectively.