Name: isolation of exosomes from the plasma of hiv-1 positive individuals
Uploaded: 2016-01-05T15:00:01
Description: Watch this Scientific Journal Video about Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals at JoVE.com

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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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.

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	<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. ...

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

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' ...

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, ...

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 ...

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 ...

In vitro Uncoating of HIV-1 Cores

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 ...

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 ...

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 ...

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

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 ...

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

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 ...

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 ...