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 writte...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+treatment+during+acute+simian+immunodeficiency+virus+(SIV)+infection+increases+viral+set+point+and+accelerates+disease+progression+despite+the+induction+of+stronger+SIV-specific+CD8++T+cell+responses.">IL-15 treatment during acute simian immunodeficiency virus (SIV) infection increases viral set point and accelerates disease progression despite the induction of stronger SIV-specific CD8+ T cell responses.</a> J Immunol. 180, 350-360 (2008).</li><li>O'Brien, M., Manches, O., Bhardwaj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmacytoid+dendritic+cells+in+HIV+infection.">Plasmacytoid dendritic cells in HIV infection.</a> Adv Exp Med Biol. 762, 71-107 (2013).</li><li>Chihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+proteins+preferentially+activate+anti-inflammatory+M2-type+macrophages.">HIV-1 proteins preferentially activate anti-inflammatory M2-type macrophages.</a> J Immunol. 188, 3620-3627 (2012).</li><li>Johnstone, R. 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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...

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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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 ...

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

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. ...

During HIV infection, both viruses and microvesicles called exosomes are produced by virus-infected cells. The overall goal of this procedure is to separate exosomes from the virus particles in plasma samples from HIV infected patients. This method can help answer key questions in the exosome field, such as what proteins or any molecules are present in exosomes produced during HIV infection.Demonstrating the procedure will be Dr.Ming Bo Huang, instructor in the department of microbiology, biochemistry and immunology and a member of the HIV/AIDS group here at Moorehouse School of Medicine. After collecting human blood and isolating the plasma according to the text protocol, add 10 milliliters of 1X PBS to 10 milliliters of plasma in a 15 milliliter tube. Centrifuge the sample at 10, 000 times G and four degrees Celsius for 30 minutes to remove celular debris.With a sterile serological pipette, transfer the cleared plasma supernatant to a clean, 25 milliliter ultracentrifuge tube. Next, centrifuge the cleared plasma at 200, 000 times G and four degrees Celsius for two hours to separate large vesicles. Then carefully remove the supernatant and discard in the biohazard waste.Using one milliliter of 1X PBS, resuspend the pellet and incubate at room temperature for 30 minutes, swirling gently to dislodge and separate particles. Add 24 milliliters of PBS to the just resuspended exosome virus solution in a 25 milliliter ultracentrifuge tube and dinphur the tube five times to mix. Then spin again at 200, 000 times G and four degrees Celsius for two hours before discarding the PBS wash solution in the biohazard waste.To prepare six to 18%velocity gradients of iodixanol, begin with a 60%stock solution of iodixanol reagent and use 1X PBS to dilute it to 6%and 18%Turn on the stirrer and into a dual chamber gradient maker, pipette 5.5 milliliters of the 18%solution into the stirred chamber and 5.5 milliliters of the 6%solution into the reservoir chamber. Then, open the stopcock, turn on the pump and allow each solution to flow into a 14 milliliter ultracentrifuge tube. Next, carefully layer one milliliter of the exosome virus solution onto the top of each 11 milliliter gradient.Then, with an SW 40 Ti swinging bucket rotor, centrifuge the gradients at 250, 000 times G and four degrees Celsius for two hours. In the meantime, label 12 1.5 milliliter microcentrifuge tubes. After the spin, remove one milliliter from the top of the gradient and transfer to tube number one.Then transfer the remaining one milliliter fractions to tubes two through 12 in sequential order. Make the assay reagent by mixing 100 parts of 1X PBS, two parts of substrate and five parts of color indicator. Next, transfer 50 microliters in duplicate of each of the 12 one milliliter gradient fractions to the wells of a 96 well microtiter plate.Prepare a set of standards by first making a 2, 000 milliunits per milliliter ACHE stock solution in PBS. Then, make 11 two-fold serial dilutions of the stock and add 50 microliters of each dilution beginning with 2, 000 milliunits per milliliter to a single well of a 96 well microtiter plate. Add 200 microliters of assay reagent mixture to each well of standards and gradient fractions and incubate in a plate reader for 20 minutes to allow for color development.Finally, using a fluorescence microplate reader, measure ACHE activity at a wavelength of 450 nanometers. Carry out additional assays according to the text protocol. As shown here, isolated exosomes identified by ACHE activity segregated in lower density fractions one to three at the top of the iodixanol gradients, whereas virus particles identified by HIV antigen P24 segregated in the higher density fractions 10 through 12.This table shows the analysis of exosomes and unfractionated plasma for 21 cytokines and chemokines using a multiplex assay. As indicated here, all 21 cytokines and chemokines were detected in exosomes from HIV-1 positive individuals and their levels were significantly elevated as compared to plasma and exosomes from HIV-1 zero one negative controls. In this figure, peripheral blood mononuclear cells or PBMCs from uninfected donors were exposed to pooled exosomes from HIV-1 positive or negative individuals.By 48 hours post-exposure, levels of the activation mark, CD38, on the surface of naive and central memory CD4 positive and CD8 positive t-cells were significantly elevated if they had been exposed to HIV-1 positive exosomes compared to HIV negative treatment. During the acetylcholinesterase assay, it's important to remember to shield the plate from strong light. This technique has paved the way for researchers in the field of HIV/AIDS to explore the role of exosomes in HIV infection and in immune activation.

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17.2K visualizações.  Morehouse School of Medicine. During HIV infection, both viruses and microvesicles called exosomes are produced by virus-infected cells. The overall goal of this procedure is to separate exosomes from the virus particles in plasma samples from HIV infected patients. This method can help answer key questions in the exosome field, such as what proteins or any molecules are present in exosomes produced during HIV infection.Demonstrating the procedure will be Dr.Ming Bo Huang, instructor in the department of microbiolo...