Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

A general diagram of the exosome isolation and purification procedure is provided in Figure 1. Whole blood was obtained from healthy volunteer donors and from HIV-positive individuals not receiving antiretroviral theraoy attending the Hope Clinic of Emory University and the Infectious Disease Program of Grady Health System in Atlanta, Georgia. This study was approved by the institutional review boards of Emory University and Morehouse School of Medicine. All persons participating in the study gave written and informed consent.

1. Preparation of Exosomes from Blood Plasma

1. Human Blood Collection and Processing
   1. Collect 10 ml of peripheral blood by venipuncture in EDTA blood collection tubes (containing 18 mg potassium EDTA), and gently invert five times to mix.
   2. Centrifuge the blood collection tubes at 1,000 x g for 20 min at RT to pellet blood cells. Use a sterile pipette to transfer the plasma fraction (4-5 ml) to a 25 ml conical tube. Dilute the plasma with 10 ml of 1 X PBS. Discard blood cells (red blood cells and white cells, also known as PBMC) in an appropriately marked container for biohazard waste.
      NOTE: In the case of uninfected donor blood samples, the PBMC can be reserved for further use (see procedure IV.2, below).
   3. Store the plasma samples at 4 ^oC for short term (2-3 days) or at -80 ^oC for longer term storage.
      NOTE: Bring any frozen plasma samples to 4 ^oC before further processing.
2. Preparation of Exosome Fraction from Plasma
   1. Centrifuge plasma at 10,000 x g for 30 min at 4 °C to remove cellular debris. Use a sterile serological pipette to transfer the cleared plasma supernatant to a clean 25 ml ultracentrifuge tube. Discard the pellet in the biohazard container.
   2. Centrifuge the cleared plasma at 100,000 x g for 2 hr at 4 °C to remove large vesicles. Remove the 100,000 x g supernatant carefully by pipetting, and discard in biohazard waste. Resuspend the 100,000 x g pellet in 1 ml of 1X PBS in a clean tube and incubate at RT for 30 min, swirling gently to dislodge and separate particles.
   3. Wash the suspended 100,000 x g exosome/virus pellet by adding 25 ml of PBS. Gently invert the tube five times to mix, then centrifuge again at 100,000 x g for 2 hr at 4 °C. Discard the PBS wash solution in the biohazard waste container.
   4. Resuspend the 100,000 x g pellet in 1 ml of 1 X PBS and incubate at RT for 30 min swirling gently to dislodge and dissolve the pellet.
      NOTE: If it is not possible to proceed directly to the iodixanol gradient step, store the pellet, containing exosomes and virus particles, at 4 °C for 1-2 days until the iodixanol gradient step.

2. Purification of Exosomes

1. Generate 6%-18% velocity gradients of iodixanol using a dual-chamber gradient former apparatus.
   1. Prepare 6% and 18% solutions of the iodixanol reagent, supplied as a 60% solution in water, by dilution in PBS. Pipet 5.5 ml of the 18% solution into the stirred chamber, and pipet 5.5 ml of the 6% solution into the reservoir chamber. Turn on the stirrer, open the stopcock, and allow each gradient to flow into a 14 ml ultracentrifuge tube.
      NOTE: Either use immediately or store prepared gradients O/N at 4 °C prior to use.
   2. Carefully layer 1 ml of the exosome/virus solution onto the top of each 11 ml gradient. Centrifuge gradients at 250,000 x g for 2 hr at 4 °C, using a SW40Ti swinging bucket rotor.
   3. Label twelve (12) 1.5 ml microcentrifuge tubes. Remove 1.0 ml from the top of the gradient and transfer to tube #1. Transfer the remaining 1ml fractions to tubes 2-12 in sequential order.
      NOTE: The topmost fraction will thus be #1, the bottom fraction, #12. Store the gradient fractions at 4 °C. The purified exosomes, which should be in the fractions 1-3 at the top of the gradient, are stable for 3-4 weeks when stored at 4 °C.

3. Exosome Characterization

1. Acetylcholinesterase (AChE) Activity Assay
   1. Prepare 100 mM substrate stock by mixing acetylthiocholine iodide 28.9 mg in 1 ml of 1X PBS. Store substrate stock at -20 °C up to 1 month.
   2. Prepare 10 mM color indicator stock by mixing benzoic acid 39.6mg and sodium bicarbonate 15 mg in 10 ml of 1X PBS. Store color indicator stock at 4 °C up to two weeks.
   3. Prepare assay reagent by mixing 1X PBS, substrate, and color indicator in a ratio of 100:2:5 (for example, 10 ml 1X PBS + 200 μl substrate + 500 μl color indicator).
   4. Transfer 50 μl from each 1.5 ml gradient fraction tube (labeled 1-12, from procedure III.3) to the wells of a 96-well microtiter plate. Prepare duplicate wells for each gradient sample.
   5. Prepare a set of standards by first making a 2000 μU/ml AChE stock in PBS. Make eleven (11) 2-fold serial dilutions of this stock and add 50 μl of each dilution to a single well of a microtiter plate, so that standard #1 = 2,000 μU/ml, #2 = 1,000 μU/ml, #3 = 500 μU/ml, etc. until #12 = 0.98 μU/ml. The twelve AchE standards will thus occupy a single row of a 96-well assay plate.
   6. Add 200 μl of assay reagent mixture to each well and incubate 20 min (in the dark) at RT to allow development of the colored reaction product. Measure AChE activity at a wavelength of 450 nm using a fluorescent microplate reader.
      NOTE: The percentage of starting material remaining after exosome purification is assessed by AchE assay of the unfractionated plasma.
2. Immunoblot Analysis
   1. Separate proteins in gradient fractions 1-12 (from procedure 2.1.3) by SDS-PAGE on 4-20% Tris-HCl precast gels at 100V x 1 hr. Transfer proteins in the gel to a nitrocellulose membrane using an electro-blotting transfer cell according to the manufacturer's instructions. Allow the transfers to blot for 12-16 hr (O/N) at 350V.
   2. Remove the membrane from the blotting apparatus and wash in Tris-Buffered Saline (TBS) for 5 min. Block with 5% nonfat milk in TTBS (TBS with 0.1% Tween 20) for 1 hr by shaking at RT.
   3. Incubate the membrane with primary antibodies specific for exosomes (CD9, CD45, CD63) or HIV-1 capsid protein (p24) in 5% nonfat milk with shaking at 4 °C for 12-16 hr (O/N). Dilutions of primary antibodies for immunoblots range from 1:2,000 -1:5,000.
   4. Wash the blot in TTBS for 20 min, followed by incubation with a 1: 2,000 dilution of horseradish peroxidase (HRP)-conjugated to anti-IgG (secondary antibody), in 5% nonfat milk for 1 hr at RT. Wash the blot 3 times with TTBS, 10 min per wash.
   5. Incubate the blot with luminol substrate reagent according to the manufacturer's directions, to generate a chemiluminescent signal from the HRP-labeled proteins Detect the chemiluminescent signal using an electronic imaging system with CCD camera.
   6. Save the images as TIFF files which can be viewed in Adobe Photoshop. Perform densitometry analysis of bands using ImageJ software (National Institutes of Health, Bethesda, MD).
3. Cytokine Assay
   1. Prior to the cytokine assay, disrupt immune complexes that are usually present in human plasma by acid dissociation and lyse the exosomes by detergent treatment. Assay both starting plasma samples and purified exosome preparations.
      1. To 100 μl of human plasma add 100 μl 0.33 N HCL and incubate at 37 °C for 1 hr. Add 100 μl 0.33 N NaOH to neutralize acid-treated plasma. Add Triton X-100 to both the neutralized plasma and purified exosome samples, to a final concentration of 1%, to cause lysis of exosomes.
   2. For this assay (a fluorescent bead-based procedure), use magnetic beads pre-coated with antibodies by the manufacturer. Use a custom-designed panel of anti-human cytokine antibody-coated beads; listed in Table 1. Vortex the antibody-coated magnetic beads from the cytokine assay kit for 30 sec and add 50 μl beads to each well of a 96-well plate to be used in the assay.
   3. Prepare a dilution series of standards in plasma standard buffer (both contained in the cytokine assay kit) as described in the instruction manual for the cytokine assay kit, to generate an 8-point standard curve.
   4. Add 150 μl of 1X wash buffer (from kit) to each well and wash the beads using a magnetic bead washer, as described in the manual. Add 25 μl plasma assay buffer (from kit) to each well. Add 25 μl of standards and samples to all wells used in the assay. Add 25 μl of plasma sample buffer to negative control wells. Seal and shake the plate at 700 rpm for 60 min at RT and incubate O/N at 4 °C.
   5. Add 150 μl of 1X wash buffer to each well and wash the plate (as in step 3.3.4, above). Add 25 μl of detection antibodies into each well, seal and shake plate at 700 rpm for 30 min at RT. Repeat the washing step.
   6. Add 50 µl of streptavidin solution (SAPE, from kit) into each well, seal and shake plate at 700 rpm for 30 min at RT. Repeat plate washes as in 3.3.4.
   7. Add 120 µl of reading buffer (from kit) into each well and shake on a laboratory tabletop shaker at 700 rpm for 5 min at RT to allow the fluorescent signal to develop. Read plate on the plate reader as per the manufacturer's instructions.
   8. In order to account for the amount of exosomes lost during the isolation procedure, use the following equation: [(original reading / plasma volume) x 66.6= adjusted cytokine reading].

4. Assay for Immunomodulatory Potential

1. Preparation of Culture Medium with Exosome-depleted Fetal Bovine Serum
   1. Centrifuge 500 ml of fetal bovine serum (FBS) at 100,000 x g for 20 hr at 4 °C to pellet contaminating exosomes and microvesicles. Carefully remove and save the exosome-depleted FBS supernatant. Discard the pellet in the biohazard waste.
   2. Combine 20% of the exosome-depleted FBS supernatant with 500 ml of Roswell Park Memorial Institute 1640 (RPMI 1640) medium. Filter the medium through a 0.45 µm membrane filter.
   3. Add streptomycin (100 U/ml), penicillin (100 U/ml), L-glutamine (2 mM), HEPES-buffered saline solution (10 µM), and IL-2 (20 U/ml) to the filtered medium.
2. Cell Culture and Exosome Exposure
   1. Expose exosomes to donor peripheral blood mononuclear cells (PBMC) obtained from healthy clinic volunteers. Suspend PBMC in prepared culture medium and count the cells using a cell/particle counter (according to the manufacturer's standard method).
      NOTE: The PBMC are obtained during the plasma preparation described above (step 1.2).
   2. Using prepared medium from procedure 4.1.3, co-culture 3.0 x 10⁶ (PBMC) with 1 μg/ml of pooled exosomes from three (3) HIV-1 seropositive or from three (3) HIV-1 seronegative individuals in a total volume of 1 ml in the wells of a 12-well culture plate (with lid). Incubate cultures for 48 hr at 37 °C.
   3. Prepare untreated PBMC cultures and cultures treated with Concanavalin A (Con A; 5 µg/ml) to serve as negative and positive controls, respectively, as described above (4.2.2). Incubate all PBMC cultures for 48 hr at 37 °C.
   4. Immediately prior to harvesting the cells, prepare dilutions of the following fluorochrome-conjugated monoclonal antibodies: Alexa Fluor 700-labeled anti-CD3 (1:400 dilution), allophycocyanin (APC)/cyanine 7 (Cy7)-labeled anti-CD4 (1:400), peridinin chlorophyll protein complex-labeled anti-CD4 (1:400), V450-labeled anti-CD8 (1:400), biotin-labeled anti-CD45RA (1:1,000), phycoerythrin (PE)/Cy7-labeled anti-CD62L (1:1,000), PE/cyanine 5 (Cy5)-labeled anti-CD38 (1:200), PE-Texas Red-labeled anti-streptavidin (1:2,000), and PE/Cy5-labeled mouse IgG1K isotype control (1:200).
   5. After the 48 hr incubation period, harvest the PBMC. Wash PBMC in PBS to remove exosomes and stain the cells by incubation for 1hr at 4 °C with individual fluorochrome-tagged antibodies. Analyze the stained PBMC by flow cytometry to quantify the expression of chemokines (as described ²¹).