Name: conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay
Uploaded: 2014-09-14T15:00:00
Description: Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

We thank Dr. James Robinson for his generous gift of A32, 17b, 48d, and C11 mAbs. PGT 121 was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (Cat#12343). This work was supported by a Canada Foundation for Innovation Program Leader #29866, by a CIHR operating #257792, by a FRQS Establishment of Young Scientist grant #24639 to AF and by a CRCHUM continuum grant as well as by a CIHR catalyst grant #126630 to AF and MR. AF is the recipient of a FRSQ Chercheur Boursier Junior 1 fellowship #24639. MV was supported by a CIHR Doctoral Research Award #291485.

1. Day 1 &#8211; Cell Culture
<ol>
	<li>Plate 2 x&#160;104 human osteosarcoma (HOS) cells per well in an opaque, 96-well cell-culture plate suitable for luminescence reading.&#160;Use Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Incubate until next day at 37 &#176;C, 5% CO2.</li>
</ol>
2. Day 2 &#8211; Polyethylenimine (PEI) Transfection
<ol>
	<li>Prepare transfection mix according to su...

<ol>
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R., 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=Immunization+with+envelope+subunit+vaccine+products+elicits+neutralizing+antibodies+against+laboratory-adapted+but+not+primary+isolates+of+human+immunodeficiency+virus+type+1.">Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1.</a> The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation. 173, 340-348 (1996).</li><li>Rerks-Ngarm, S., 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=Vaccination+with+ALVAC+and+AIDSVAX+to+prevent+HIV-1+infection+in+Thailand.">Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.</a> N. Engl. J. 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Virol. 65, 2119-2123 (1991).</li><li>Haim, H., Salas, I., Sodroski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+of+the+human+immunodeficiency+virus+envelope+glycoprotein+precursor+decreases+conformational+flexibility.">Proteolytic processing of the human immunodeficiency virus envelope glycoprotein precursor decreases conformational flexibility.</a> J. Virol. 87, 1884-1889 (2013).</li><li>Chen, L., 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=Structural+basis+of+immune+evasion+at+the+site+of+CD4+attachment+on+HIV-1+gp120.">Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120.</a> Science. 326, 1123-1127 (2009).</li><li>Kwong, P. 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Immunol. 43, 3087-3089 (2013).</li><li>Veillette, 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=Interaction+with+Cellular+CD4+Exposes+HIV-1+Envelope+Epitopes+Targeted+by+Antibody-Dependent+Cell-Mediated+Cytotoxicity.">Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity.</a> J. Virol. 88, 2633-2644 (2014).</li><li>Wibmer, C. K., 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=Viral+escape+from+HIV-1+neutralizing+antibodies+drives+increased+plasma+neutralization+breadth+through+sequential+recognition+of+multiple+epitopes+and+immunotypes.">Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.</a> PLoS Pathogens. 9 (e1003738), (2013).</li><li>Kovacs, J. 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=HIV-1+envelope+trimer+elicits+more+potent+neutralizing+antibody+responses+than+monomeric+gp120.">HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120.</a> Proc. Natl. Acad. Sci. 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Virol. 87, 2549-2562 (2013).</li><li>Haim, H., 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=Soluble+CD4+and+CD4-mimetic+compounds+inhibit+HIV-1+infection+by+induction+of+a+short-lived+activated+state.">Soluble CD4 and CD4-mimetic compounds inhibit HIV-1 infection by induction of a short-lived activated state.</a> PLoS Pathogens. 5 (e1000360), (2009).</li><li>Haim, H., 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=Contribution+of+intrinsic+reactivity+of+the+HIV-1+envelope+glycoproteins+to+CD4-independent+infection+and+global+inhibitor+sensitivity.">Contribution of intrinsic reactivity of the HIV-1 envelope glycoproteins to CD4-independent infection and global inhibitor sensitivity.</a> PLoS Pathogens. 7 (e1002101), (2011).</li><li>Thali, 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=Effects+of+changes+in+gp120-CD4+binding+affinity+on+human+immunodeficiency+virus+type+1+envelope+glycoprotein+function+and+soluble+CD4+sensitivity.">Effects of changes in gp120-CD4 binding affinity on human immunodeficiency virus type 1 envelope glycoprotein function and soluble CD4 sensitivity.</a> J. 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This assay is optimized to detect the interaction of specific mAbs with HIV-1 trimeric Env expressed at the cell surface. Once the protocol has been established, it can be used at medium to high throughputs with low overall material costs and little amounts of antibodies. Since this assay is transfection-based, it can easily be adapted for coexpression of cellular proteins such as CD4 in order to study their effects on Env conformation.
However, the transfection base of this protocol also impl...

Human immunodeficiency virus type 1 (HIV-1) entry, mediated by the trimeric viral envelope glycoproteins (Env) is the first step of the infectious cycle. Being the only exposed viral antigen presented at the surface of virions, the Env trimer elicits neutralizing and nonneutralizing antibodies. As such, it represents an interesting candidate for vaccine immunogen design. However, vaccination trials with Env in soluble or recombinant forms elicited responses with only minimal effectiveness against most primary HIV-1 isolates1-3. Nonetheless, partial efficacy observed in the RV144 vaccine trial4 renewed interest in HIV-1 Env as an immunogen candidate. This was corroborated by a recent study describing that vaccine-elicited anti-Env antibodies were sufficient to generate a certain degree of protection against SIV and HIV challenges5.
After being synthesized in the endoplasmic reticulum, the Env glycoprotein precursor, gp160, undergoes various post-translational modifications that are critical for its ability to fuel the viral fusion process. The Env precursor must fold properly and associate in trimers before being cleaved into its extra-cytoplasmic gp120 and transmembrane gp41 subunits6-10, with noncovalent interactions maintaining the gp120-gp41 liaison. The infected cell machinery is also responsible for heavily glycosylating Env, comprising about 50% of its total mass11,12. The resulting complex structure allows Env to be conformationally flexible13,14, while providing a metastability that is thought to allow Env to adapt and hide certain highly immunogenic epitopes that would otherwise be exposed15-19, highlighting the importance to better understand the different conformations sampled by the native Env trimer.
To date, several techniques have been developed and successfully used to study Env conformational changes. However, they vary in their limitations, being often restricted to specific Env contexts. For example, surface plasmon resonance or immunoprecipitation assays using conformation specific monoclonal antibodies (mAbs), rely either on monomeric soluble or solubilized Env molecules which are known to be immunogenetically different from their trimeric forms20,21. Recent studies also suggest that cleavage affects Env conformations resulting in the exposure of epitopes mainly recognized by nonneutralizing antibodies14,22,23.
Here we describe in detail a method that allows for fast and easy determination of the conformation of cellularly-expressed Env trimers18,24-26. Following transient transfection of Env in a human adherent cell line the binding of Env-specific antibodies is detected using a simple chemiluminescence reaction. This technique can also be used to characterize the conformational preference of conformation-dependent antibodies. Thus, this assay provides a robust and highly flexible detection method.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOS cells</td>
			<td style="width:103px;">ATCC</td>
			<td style="width:119px;">CRL-1543</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">White Opaque Tissue Culture Plate, 96 well, Flat Bottom</td>
			<td style="width:103px;">BD</td>
			<td align="right" style="width:119px;">353296</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyethylenimine, linear, 25000 MW</td>
			<td style="width:103px;">Polysciences</td>
			<td align="right" style="width:119px;">23966</td>
			<td>Prepared in 1mg/ml solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:103px;">Invitrogen</td>
			<td align="right" style="width:119px;">11995</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Goat Anti-Human IgG, Peroxidase Conjugated</td>
			<td style="width:103px;">Pierce</td>
			<td align="right" style="width:119px;">31413</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Enhanced Chemiluminescence Substrate</td>
			<td style="width:103px;">PerkinElmer</td>
			<td style="width:119px;">NEL105001EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TriStar LB 941, Plate Reader</td>
			<td style="width:103px;">Berthold Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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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.

Using the general procedure described above, we adapted the protocol to assay the impact of soluble CD4 (sCD4) and coexpressed cellular CD4 on the exposure of CD4i epitopes on either wild-type (wt) or mutated Env, as described previously18,24,25,28. Figure 1 schematically represents the general procedure and the exposure of CD4i epitopes following treatment with sCD4 or by coexpression of cellular CD418. In Figure 2, we used sCD4 to induce Env conformational changes...

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

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 ...

Research

JoVE Journal

Immunology and Infection

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In vitro Uncoating of HIV-1 Cores

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Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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

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