We thank the following people: Jane Chu, Cameron Tran, James Lillard, Mafuz Khan, Masebonang Albert, Ken Rogers, and Syed A. Ali. Kateena Addae-Konadu was supported by UNCF/Merck Graduate Research Fellowship, American Medical Association Foundation, CRECD Grant 8R25MD007589-10, and NIH NIGMS MBRS Grant R25 GM058268. This work was supported by NIMHD grants 8G12MD007602, and 8U54MD007588, NIAID grant 1R21AI095150-01A1, Georgia Research Alliance grant GRA.VAC08.W, and Emory CFAR grant P30 A1050409.

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
<ol>
	<li>Human Blood Collection and Processing
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of Exosome Fraction from Plasma
	<ol>
		<li>Centrifuge plasma at 10,000 x g for 30 min at 4 &#176;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.</li>
		<li>Centrifuge the cleared plasma at 100,000 x g for 2 hr at 4 &#176;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.</li>
		<li>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 &#176;C. Discard the PBS wash solution in the biohazard waste container.</li>
		<li>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 &#176;C for 1-2 days until the iodixanol gradient step.</li>
	</ol>
	</li>
</ol>
2. Purification of Exosomes
<ol>
	<li>Generate 6%-18% velocity gradients of iodixanol using a dual-chamber gradient former apparatus.
	<ol>
		<li>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 &#176;C prior to use.</li>
		<li>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 &#176;C, using a SW40Ti swinging bucket rotor.</li>
		<li>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 &#176;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 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Exosome Characterization
<ol>
	<li>Acetylcholinesterase (AChE) Activity Assay
	<ol>
		<li>Prepare 100 mM substrate stock by mixing acetylthiocholine iodide 28.9 mg in 1 ml of 1X PBS. Store substrate stock at -20 &#176;C up to 1 month.</li>
		<li>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 &#176;C up to two weeks.</li>
		<li>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 &#956;l substrate + 500 &#956;l color indicator).</li>
		<li>Transfer 50 &#956;l 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.</li>
		<li>Prepare a set of standards by first making a 2000 &#956;U/ml AChE stock in PBS. Make eleven (11) 2-fold serial dilutions of this stock and add 50 &#956;l of each dilution to a single well of a microtiter plate, so that standard #1 = 2,000 &#956;U/ml, #2 = 1,000 &#956;U/ml, #3 = 500 &#956;U/ml, etc. until #12 = 0.98 &#956;U/ml. The twelve AchE standards will thus occupy a single row of a 96-well assay plate.</li>
		<li>Add 200 &#956;l 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.</li>
	</ol>
	</li>
	<li>Immunoblot Analysis
	<ol>
		<li>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&#39;s instructions. Allow the transfers to blot for 12-16 hr (O/N) at 350V.</li>
		<li>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.</li>
		<li>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 &#176;C for 12-16 hr (O/N). Dilutions of primary antibodies for immunoblots range from 1:2,000 -1:5,000.</li>
		<li>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.</li>
		<li>Incubate the blot with luminol substrate reagent according to the manufacturer&#39;s directions, to generate a chemiluminescent signal from the HRP-labeled proteins Detect the chemiluminescent signal using an electronic imaging system with CCD camera.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Cytokine Assay
	<ol>
		<li>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.
		<ol>
			<li>To 100 &#956;l of human plasma add 100 &#956;l 0.33 N HCL and incubate at 37 &#176;C for 1 hr. Add 100 &#956;l 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.</li>
		</ol>
		</li>
		<li>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 &#956;l beads to each well of a 96-well plate to be used in the assay.</li>
		<li>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.</li>
		<li>Add 150 &#956;l 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 &#956;l plasma assay buffer (from kit) to each well. Add 25 &#956;l of standards and samples to all wells used in the assay. Add 25 &#956;l 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 &#176;C.</li>
		<li>Add 150 &#956;l of 1X wash buffer to each well and wash the plate (as in step 3.3.4, above). Add 25 &#956;l of detection antibodies into each well, seal and shake plate at 700 rpm for 30 min at RT. Repeat the washing step.</li>
		<li>Add 50 &#181;l 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.</li>
		<li>Add 120 &#181;l 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&#39;s instructions.</li>
		<li>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].</li>
	</ol>
	</li>
</ol>
4. Assay for Immunomodulatory Potential
<ol>
	<li>Preparation of Culture Medium with Exosome-depleted Fetal Bovine Serum
	<ol>
		<li>Centrifuge 500 ml of fetal bovine serum (FBS) at 100,000 x g for 20 hr at 4 &#176;C to pellet contaminating exosomes and microvesicles. Carefully remove and save the exosome-depleted FBS supernatant. Discard the pellet in the biohazard waste.</li>
		<li>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 &#181;m membrane filter.</li>
		<li>Add streptomycin (100 U/ml), penicillin (100 U/ml), L-glutamine (2 mM), HEPES-buffered saline solution (10 &#181;M), and IL-2 (20 U/ml) to the filtered medium.</li>
	</ol>
	</li>
	<li>Cell Culture and Exosome Exposure
	<ol>
		<li>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&#39;s standard method). 
		NOTE: The PBMC are obtained during the plasma preparation described above (step 1.2).</li>
		<li>Using prepared medium from procedure 4.1.3, co-culture 3.0 x 106 (PBMC) with 1 &#956;g/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 &#176;C.</li>
		<li>Prepare untreated PBMC cultures and cultures treated with Concanavalin A (Con A; 5 &#181;g/ml) to serve as negative and positive controls, respectively, as described above (4.2.2). Incubate all PBMC cultures for 48 hr at 37 &#176;C.</li>
		<li>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).</li>
		<li>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 &#176;C with individual fluorochrome-tagged antibodies. Analyze the stained PBMC by flow cytometry to quantify the expression of chemokines (as described 21).</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Chronic immune activation (CIA) and CD4+ T cell depletion are two important hallmarks of HIV-1 infection. They have been established as predictors for pathogenesis, with CIA being the best predictor. However, the underlying mechanisms driving chronic systemic immune activation and CD4+ T cell decline still have not been fully elucidated. We and other labs have developed firm evidence that exosomes secreted from HIV-1 infected cells play a role in both hallmarks.
The continuing interest in both composition and function of extracellular vesicles has led to the publication of various methods for exosome isolation from both cell culture media and biological fluids24-26. However, a barrier to investigating the role of exosomes in HIV-1 pathogenesis has been the efficient separation of exosomes from HIV-1 particles while maintaining the ability to investigate both exosome content and functional activity. We have developed a protocol for purification of exosomes from HIV-1 particles in human plasma, utilizing iodixanol velocity gradients. The HIV-positive donors used in this study had not received antiretroviral treatment and the plasma samples used for these experiments contained from 1500 to 400,000 virus particles/mL with an average of 206,000 virus particles/ml21. Thus, we demonstrate that exosomes in the plasma of HIV-1 infected individuals can be efficiently separated from HIV-1 virus particles, even when the virus loads are high. Though similar in size and density, exosomes segregated in the low-density/upper fractions of the iodixanol gradients as compared to viral particles, which segregated in the high-density/lower fractions. The exosomes prepared by the iodixanol gradient method are highly purified and free of contaminating extracellular proteins. The purity of the isolated exosomes population was confirmed using p24 ELISA and Western blot analysis for HIV-1 p24 capsid protein as well as Western analysis for exosomal markers, AChE, CD9, CD45, and CD63. Use of these markers is consistent with recently published guidelines for exosome identification27.
More physiologically relevant to immune activation, the purified exosomes from HIV-1 infected individuals were found to contain cytokines/chemokines at significantly higher concentrations than exosomes from HIV-1 seronegative controls. Moreover, exosomes from HIV-1 infected individuals were biologically active, exhibiting the ability to induce increased levels of the activation marker, CD38, on the surface of na&#239;ve and central memory CD4+ and CD8 + T cells. Collectively, these data suggest a mechanism that could drive persistent immune activation during HIV infection.
The results we were able to obtain using our exosome purification process, which combines differential centrifugation and iodixanol velocity gradient separation show the value of using highly purified exosomes for physiological and functional bioassays. The procedure does have certain pitfalls and limitations. A considerable amount of the exosomes, often a majority of the starting material as calculated by measurements of AChE activity, are lost during the process. In addition, the procedure is also time consuming and requires access to expensive equipment. Finally, the generation of the iodixanol gradients using the gradient fromer apparatus requires considerable practice to ensure reproducibility. An alternative method for gradient generation might be to prepare a graded series of iodixanol solutions, followed by careful layering of the solutions into centrifuge tubes and incubating O/N to allow the gradients to form. However, we have not tested this alternative.
The experimental protocol described here for isolation and separation of exosomes from HIV-1 particles in human plasma is an effective method to ensure the purity of exosomes. Use of this method has led to exciting avenues for future inquiry regarding the role of exosomes in HIV-1 pathogenesis and could equally be used in inquiries for other biological processes.

The HIV-1 epidemic continues to have a significant impact throughout the world. As of 2013, approximately 35 million people worldwide were living with HIV, and 2.1 million of these were newly infected individuals1. Prevention strategies and increased access to antiretroviral therapy have been helpful in reducing the overall acquisition of HIV. However, individual populations are still experiencing rises in the acquisition of HIV1. Thus, there is a need for continued efforts to address this epidemic.
One of the strongest predictors of HIV disease progression is chronic immune activation (CIA)2-10. Defined by persistently high levels of detectable cytokines and elevated expression markers on the surface of T lymphocytes, CIA has been attributed to: i) continuous dendritic cell production of Type I IFN11; (ii) direct immune activation driven by HIV proteins Tat, Nef and gp12012; (iii) translocation of bacterial proteins in gut associated immune cells6. However, the exact mechanism(s) underlying chronic, systemic immune activation in HIV infection remain to be fully elucidated.
Our research group and others have demonstrated a role of exosomes in HIV pathogenesis15-18. Our group has determined that the Nef protein is excreted from infected cells in exosomes15, and exosomal Nef (exNef) is present in the plasma of HIV-infected individuals at nanogram levels18. We have shown that bystander CD4+ T-cells exposed to exNef resulted in activation-induced cell death dependent on the CXCR4 pathway19, 20. Alternatively, monocyte/macrophages were refractory to exNef-induced apoptosis, but exhibited altered cellular functions and cytokine expression. Most recently, our group has shown exosomes isolated from the plasma of HIV-infected individuals contain a variety of pro-inflammatory cytokines. Further, na&#239;ve peripheral blood mononuclear cells exposed to plasma-derived exosomes from HIV-infected patients induced expression of CD38 on na&#239;ve and central memory CD4+ and CD8+ T cells. This likely contributes to systemic inflammation and viral propagation via bystander cell activation21, and suggests that exosomes play a significant role in HIV pathogenesis.
In investigating the role of exosomes in HIV pathogenesis, one challenge is developing techniques to efficiently separate exosomes from HIV particles while maintaining the exosomal content as well as their functional immune modulatory capability. Several methods have been described for isolating exosomes from cell culture and biological fluids22,23. Because of their small size and low density (exosomes float at a density of 1.15 - 1.19 g/ml), differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation23. However, HIV-infected cell culture supernatants and patients&#39; plasma contain both exosomes and HIV-1 viral particles. Exosomes and HIV-1 particles are very similar in both size and density. Alternatively, taking advantage of the expression of unique exosomal markers such as CD63, CD45, and CD81, exosomes have been isolated using immunoaffinity capture methods23. This procedure can separate virus from exosomes. However, the drawback of this technique is the tight attachment of antibodies to the purified exosomes, which could interfere with assessment of the immunomodulatory potential of exosomes in culture.
To address these limitations, we developed a procedure for purification of exosomes from HIV particles in human plasma modified from Cantin and coworkers22 using Iodixanol velocity gradients. Exosomes were found to segregate in the low-density/upper fractions of iodixanol gradients, whereas virus particles segregated in the high-density/lower fractions. Virus particles were identified by p24 ELISA and exosomes were identified using exosome markers AChE, CD9, CD63, and CD45. The upper low-density fractions collected contained exosomes which were negative for HIV-1 p24 contamination. The efficient purification and separation of exosomes from HIV particles in human plasma allows for accurate examination of the content of exosomes derived from human plasma as well as the investigation of their immune modulatory potential and the diagnostic and prognostic value of exosomes in HIV-1 pathogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="506">
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">BD Vacutainer EDTA tubes (10ml)&#160;</td>
			<td style="width:92px;">Becton Dickinson</td>
			<td style="width:101px;">368589</td>
			<td style="width:127px;">pink top tubes</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Lymphoprep Ficoll reagent</td>
			<td style="width:92px;">Cosmo Bio</td>
			<td style="width:101px;">AXS-1114545</td>
			<td style="width:127px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Optiprep iodixanol reagent</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">D1556</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">14ml ultracentrifuge tubes</td>
			<td style="width:92px;">Beckman Coulter</td>
			<td style="width:101px;">344060</td>
			<td style="width:127px;">ultraclear tubes</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Gradient Former Model 485</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">165-4120</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Acetylthiocholine iodide</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">1480</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Benzoic Acid</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">D8130</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Sodium Bicarbonate</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">S5761</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Acetyl Cholinesterase</td>
			<td style="width:92px;">Sigma</td>
			<td style="width:101px;">C3389</td>
			<td style="width:127px;"></td>
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		<tr height="58">
			<td height="58" style="height:58px;width:187px;">96-well clear microtiter plate</td>
			<td style="width:92px;">Medical Supply Partners</td>
			<td style="width:101px;">TR5003</td>
			<td style="width:127px;"></td>
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		<tr height="39">
			<td height="39" style="height:39px;width:187px;">SpectraMax 190 microplate reader</td>
			<td style="width:92px;">Molecular Devices</td>
			<td style="width:101px;">190</td>
			<td style="width:127px;">Fluorescent plate reader</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Criterion Gel Electrophoresis Cell</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">165-6001</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Transblot Gel&#160;</td>
			<td rowspan="2" style="width:92px;">BIO-RAD</td>
			<td rowspan="2" style="width:101px;">170-3910</td>
			<td rowspan="2" style="width:127px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Transfer Cell</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Tris-HCl Criterion precast gels</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">567-1093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Anti-CD45 antibody&#160;</td>
			<td style="width:92px;">Abcam&#160;</td>
			<td style="width:101px;">Ab10558</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">CD63 Antibody (H-193)</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-15363</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">CD9 Antibody (H-110)</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-9148</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Rabbit pAb p24 HIV-1&#160;</td>
			<td style="width:92px;">ImmunoDX, LLC</td>
			<td style="width:101px;">1303</td>
			<td style="width:127px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Nitrocellulose membrane&#160;</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">G1472430</td>
			<td style="width:127px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Tris Buffered Saline</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">170-6435</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">HRP-conjugated IgG (H+L) secondary antibody</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:101px;">31460</td>
			<td style="width:127px;">Goat-Anti-Rabbit</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">HRP-conjugated IgG (H+L) secondary antibody</td>
			<td style="width:92px;">Thermo Scientific</td>
			<td style="width:101px;">31430</td>
			<td style="width:127px;">Goat-Anti-Mouse</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Western Blotting Luminol Reagent</td>
			<td style="width:92px;">Santa Cruz Biotech, Inc.</td>
			<td style="width:101px;">SC-2048</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">GE LAS-4010 Imager</td>
			<td style="width:92px;">GE Healthcare</td>
			<td style="width:101px;">LAS-4010</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Human Procarta Cytokine Immunoassay Kit</td>
			<td style="width:92px;">Affymetrix&#160;</td>
			<td style="width:101px;">N/A</td>
			<td style="width:127px;">Custom immunoassay panel</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Bio-Plex 200 Immunobead Reader</td>
			<td style="width:92px;">BIO-RAD</td>
			<td style="width:101px;">171-000201</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">Coulter Z2 Particle Counter</td>
			<td style="width:92px;">Beckman Coulter</td>
			<td style="width:101px;">383552</td>
			<td style="width:127px;">Cell counter</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Alexa Fluor 700-labeled anti-CD3</td>
			<td style="width:92px;">BD Bioscience (UCHT1)</td>
			<td style="width:101px;">300424</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled anti-CD4</td>
			<td style="width:92px;">Biolegend (OKT4)</td>
			<td style="width:101px;">317418</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">PerCP-labeled anti-CD4</td>
			<td style="width:92px;">BD Bioscience (RPA-T8)</td>
			<td style="width:101px;">550631</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">V450-labeled anti-CD8</td>
			<td style="width:92px;">BD Bioscience (RPA-T8)</td>
			<td style="width:101px;">560347</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:187px;">Biotin-labeled anti-CD45RA</td>
			<td style="width:92px;">BD Bioscience (HI100)</td>
			<td style="width:101px;">555487</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy7-labeled anti-CD62L</td>
			<td style="width:92px;">Biolegend (DREG-56)</td>
			<td style="width:101px;">304822</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy5-labeled anti-CD38</td>
			<td style="width:92px;">Biolegend (HIT2)</td>
			<td style="width:101px;">303508</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled anti-HLADR</td>
			<td style="width:92px;">Biolegend (L243)</td>
			<td style="width:101px;">307618</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE-Texas Red-labeled anti-streptavidin</td>
			<td style="width:92px;">BD Bioscience</td>
			<td style="width:101px;">551487</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">PE/Cy5-labeled mouse IgG1K</td>
			<td style="width:92px;">Biolegend (MOPC-21)</td>
			<td style="width:101px;">400116</td>
			<td style="width:127px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:187px;">APC/Cy7-labeled mouse IgG2aK</td>
			<td style="width:92px;">Biolegend (MOPC-173)</td>
			<td style="width:101px;">400229</td>
			<td style="width:127px;"></td>
		</tr>
	</tbody>
</table>

isolation of exosomes from the plasma of hiv-1 positive individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Exosomes are efficiently purified from HIV-1 positive human plasma. Isolated exosomes, identified by acetylcholinesterase (AChE) activity, segregated in lower density fractions 1-3 at the top of the iodixanol gradients, whereas virus particles, identified by HIV-1 antigen p24, segregated in the higher-density fractions (10-12, near the bottom). The presence of exosomes was further confirmed by immunoblot identification of exosome markers AChE, CD9, CD45, and CD63, and by electron microscopy (Figure 2).
Pro-inflammatory cytokines and chemokines are associated with and significantly elevated in the exosomes of HIV-1 seropositive individuals. Purified exosomes and unfractionated plasma from HIV-1 infected and HIV-1 seronegative individuals were analyzed for 21 cytokines/chemokines by multiplex assay. All 21 cytokines/chemokines were detected in exosomes isolated from HIV-1 positive individuals. Additionally, their levels were significantly elevated as compared to plasma and exosomes from HIV-1 seronegative controls (Table 1).
CD38 expression was increased on the surface of cells exposed to exosomes from HIV-1 seropositive individuals. PBMCs from uninfected human donors were exposed for 48 hr to pooled exosomes from HIV-1 seropositive or seronegative individuals and assessed for levels of the activation marker CD38 on CD4+ and CD8+ T-cells via flow cytometry. We observed that by 48 hr post-exposure, CD38 expression on the surface of na&#239;ve and central memory CD4+ and CD8+ T cells were significantly elevated in T-cells exposed to exosomes from HIV-1 positive individuals compared to HIV-negative exosome treatment and untreated controls (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/53495/53495fig1.jpg" /> 
Figure 1. Schematic representation of exosome isolation from HIV-1 positive human plasma. (A) 10 ml of peripheral blood was collected from HIV-1 seropositve and seronegative individuals. (B) EDTA blood collection tubes were centrifuged at 1,000 x g for 20 min at RT. (C) Separated plasma was transferred to 50 ml conical tubes and diluted &#189; with 1x PBS and (D) centrifuged 10,000 x g for 30 min. (E) The supernatant was transferred to ultracentrifuge tubes and (F) centrifuged at 10,000 x g for 2 hr. (G, H) The supernatant was discarded and the exosome pellet re-suspended and washed in 1 ml of 1X PBS. (I) Following centrifugation, the supernatant was discarded and the pellet re-suspended in 1ml 1x PBS and overlaid on 6-18% iodixanol velocity gradient and (J) centrifuged at 250,000 x g for 2 hr. (K) Following centrifugation, 1 ml fractions from top to bottom of the gradient were transferred to 1.5 ml tubes and analyzed for AChE content and immunoblot analysis. (L) Fractions 2 and 3 were than combined and diluted with 4 ml of 1X PBS and (M) centrifuged at 400,000 x g for 2 hr. (N) Following centrifugation, the supernatant was discarded and the pellet re-suspended in 1 ml of 1X PBS for analysis of pro-inflammatory cytokine and chemokine expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53495/53495fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53495/53495fig2.jpg" /> 
Figure 2. Exosomes are efficiently purified from human plasma. Individual iodixanol velocity gradient fractions from HIV-seropositive or seronegative individuals were subjected to (A) enzymatic assay for acetylcholinesterase (AChE), and (B) Western blot analysis for exosomal markers CD9, CD45, and CD63 and viral protein p24 and were (C) immunolabeled with anti-CD63 and examined with electron microscopy to confirm preparation of purified exosomes (C). (Figure from Konadu et al, 201421, used by permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53495/53495fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53495/53495fig3.jpg" /> 
Figure 3. Assay for Immunomodulatory Potential. PBMCs from HIV-1 seronegative individuals were exposed to either pooled exosomes from plasma of HIV-1 seropositive (HIV+ Exo) or seronegative (HIV- Exo) individuals, left untreated, or treated with 5 &#181;g/ml of Concanavalin A (Con A) as a positive control. After 48 hr exposure, Na&#239;ve (CD45RA+/CD62L+) , Central (TCM; CD45RA-/CD62L+) and Effector (TEM; CD45RA-/CD62L-) memory CD4+ and CD8+ T-cells were analyzed for CD38 expression by flow cytometry. Exosome concentration was normalized by total protein and added at 1 &#181;g/ml. Error bars represent mean +/- SEM of six independent donors. Difference between groups were tested for statistical significance by the One Way ANOVA Test. *p &#60; 0.05, **p&#60; 0.01,***p&#60; 0.001, ****p&#60; 0.0001. (Figure from Konadu et al, 201421,, used by permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53495/53495fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/53495/53495table1.jpg" /> 
Table 1. Analysis of purified exosomes and whole plasma from HIV-1 seropositive and seronegative individuals for pro-inflammatory cytokine and chemokine expression. Purified exosomes from HIV-1 seropositive or seronegative individuals were analyzed for pro-inflammatory cytokine and chemokine expression. All 21 pro-inflammatory cytokines and chemokines measured were associated with and were significantly elevated in exosomes of HIV-1 seropositive individuals compared to seronegative controls. Additionally, IL-1a, IFNa2, and CXCL10 were significantly elevated in the unfractionated plasma of HIV-1 seropositive individuals compared to seronegative controls. Difference between groups were tested for statistical significance by the Mann-Whitney U Test. *p &#60; 0.05, **p&#60; 0.01,***p&#60; 0.001, ****p&#60; 0.0001. (Figure from Konadu et al, 201421, used by permission). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53495/53495table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals at JoVE.com

Izolacja egzosomów z osocza osób zakażonych wirusem HIV-1 | Immunologia i zakażenia | Wideo

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

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

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17.2K Views.  Morehouse School of Medicine. Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

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

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...