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. ...

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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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 ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

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 ...