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 subsequent steps. Adjust reagents and DNA quantities according to the number of wells that are to be transfected with the same Env.</li>
	<li>Tube A: Add 10 ng Tat-encoding plasmid (such as pTat-III27) and 150 ng Env-encoding plasmid to 5 &#181;l DMEM supplemented with 25 mM HEPES.</li>
	<li>The Tat-encoding plasmid is only required when using Tat-dependent Env-encoding plasmids such as pSVIII.</li>
	<li>Tube B: Add 450 ng PEI (from a 1 &#181;g/&#181;l solution) to 5 &#181;l DMEM.</li>
	<li>Add content of tube B to tube A. Mix thoroughly by vortexing for 10 sec and incubate transfection mix 10 min at room temperature (22 &#176;C).</li>
	<li>Add 10 &#181;l of the transfection mix per well of the 96-well plate. Incubate for 48 hr at 37&#160;&#176;C, 5% CO2.</li>
</ol>
3. Day 4 &#8211; ELISA
<ol>
	<li>Perform all experiments at room temperature to minimize possible endocytosis of Env/antibodies complexes.</li>
	<li>Prepare 250 ml of Washing Buffer per plate being used at the same time. Washing Buffer is 1x Tris-buffered saline (TBS)&#160;pH 7.5 (50 mM Tris-Cl, pH 7.5; 150 mM NaCl), supplemented with 1 mM MgCl2 and 1.8&#160;mM CaCl2.</li>
	<li>Prepare 125 ml Blocking Buffer per plate by adding 1% nonfat dry milk and 5 mM Tris pH 8.0 to Washing Buffer.</li>
	<li>Remove cell culture media and transfection mix (supernatant) from 96-well plate.</li>
	<li>Add 100 &#181;l of Blocking Buffer per well and incubate 20 min at RT.</li>
	<li>Remove supernatant and add 50 &#181;l of antibody (or serum) per well, diluted to appropriate concentration in Blocking Buffer. Typically, use a concentration of 1 &#181;g/ml. Incubate 1 hr at RT.</li>
	<li>Wash 3x&#160;with 100 &#181;l Blocking Buffer and then repeat washing process 3x with 100 &#181;l Washing Buffer.</li>
	<li>Remove supernatant, add 100 &#181;l Blocking Buffer, and incubate 5 min at RT.</li>
	<li>Remove supernatant and add 50 &#181;l of secondary antibody, diluted 1/3,000 in Blocking Buffer. Vary optimal antibody dilution according to manufacturer differences. Incubate 40 min at RT.</li>
	<li>Wash 3x with 100 &#181;l Blocking Buffer and then repeat washing process 3x with 100 &#181;l Washing Buffer.</li>
</ol>
4. Data Acquisition
<ol>
	<li>Remove supernatant from the plate and add 30 &#181;l 1x&#160;enhanced chemiluminescence (ECL) substrate per well.</li>
	<li>Acquire chemiluminescence signal for 1 sec/well on a suitable plate-reader according to manufacturer instructions. Reading time may differ according to hardware differences.</li>
</ol>

<ol>
	<li>Bures, 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+recombinant+canarypox+vectors+expressing+membrane-anchored+glycoprotein+120+followed+by+glycoprotein+160+boosting+fails+to+generate+antibodies+that+neutralize+R5+primary+isolates+of+human+immunodeficiency+virus+type+1.">Immunization with recombinant canarypox vectors expressing membrane-anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1.</a> AIDS Res. Human Retroviruses. 16, 2019-2035 (2000).</li><li>Koff, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+vaccine+development:+challenges+and+opportunities+towards+solving+the+HIV+vaccine-neutralizing+antibody+problem.">HIV vaccine development: challenges and opportunities towards solving the HIV vaccine-neutralizing antibody problem.</a> Vaccine. 30, 4310-4315 (2012).</li><li>Mascola, J. 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. Med. 361, 2209-2220 (2009).</li><li>Roederer, 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=Immunological+and+virological+mechanisms+of+vaccine-mediated+protection+against+SIV+and+HIV.">Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV.</a> Nature. 505, 502-508 (2014).</li><li>Fennie, C., Lasky, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+for+intracellular+folding+of+the+human+immunodeficiency+virus+type+1+gp120.">Model for intracellular folding of the human immunodeficiency virus type 1 gp120.</a> J. Virol. 63, 639-646 (1989).</li><li>Willey, R. L., Bonifacino, J. S., Potts, B. J., Martin, M. A., Klausner, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+cleavage,+and+degradation+of+the+human+immunodeficiency+virus+1+envelope+glycoprotein+gp160.">Biosynthesis cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160.</a> Proc. Natl. Acad. Sci. U.S.A. 85, 9580-9584 (1988).</li><li>Bosch, V., Pawlita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+human+immunodeficiency+virus+type+1+env+gene+product+proteolytic+cleavage+site.">Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site.</a> J. Virol. 64, 2337-2344 (1990).</li><li>Hallenberger, 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=Inhibition+of+furin-mediated+cleavage+activation+of+HIV-1+glycoprotein+gp160.">Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.</a> Nature. 360, 358-361 (1038).</li><li>Li, Y., Luo, L., Thomas, D. Y., Kang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+Env+protein+signal+sequence+retards+its+cleavage+and+down-regulates+the+glycoprotein+folding.">The HIV-1 Env protein signal sequence retards its cleavage and down-regulates the glycoprotein folding.</a> Virology. 272, 417-428 (2000).</li><li>Leonard, 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=Assignment+of+intrachain+disulfide+bonds+and+characterization+of+potential+glycosylation+sites+of+the+type+1+recombinant+human+immunodeficiency+virus+envelope+glycoprotein+(gp120)+expressed+in+Chinese+hamster+ovary+cells.">Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.</a> J. Biol. Chem. 265, 10373-10382 (1990).</li><li>Wang, W., 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=A+systematic+study+of+the+N-glycosylation+sites+of+HIV-1+envelope+protein+on+infectivity+and+antibody-mediated+neutralization.">A systematic study of the N-glycosylation sites of HIV-1 envelope protein on infectivity and antibody-mediated neutralization.</a> Retrovirology. 10 (14), (2013).</li><li>Helseth, E., Olshevsky, U., Furman, C., 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=Human+immunodeficiency+virus+type+1+gp120+envelope+glycoprotein+regions+important+for+association+with+the+gp41+transmembrane+glycoprotein.">Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein.</a> J. 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. D., 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+evades+antibody-mediated+neutralization+through+conformational+masking+of+receptor-binding+sites.">HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.</a> Nature. 420, 678-682 (2002).</li><li>Sakai, K., Takiguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+an+effective+AIDS+vaccine+development.">Toward an effective AIDS vaccine development.</a> Eur. J. 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. U.S.A. 109, 12111-12116 (2012).</li><li>Yuan, W., Bazick, J., 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=Characterization+of+the+multiple+conformational+States+of+free+monomeric+and+trimeric+human+immunodeficiency+virus+envelope+glycoproteins+after+fixation+by+cross-linker.">Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker.</a> J. Virol. 80, 6725-6737 (2006).</li><li>Guttman, M., Lee, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+interaction+between+gp41+and+gp120+is+observed+for+monomeric+but+not+oligomeric,+uncleaved+HIV-1+Env+gp140.">A functional interaction between gp41 and gp120 is observed for monomeric but not oligomeric, uncleaved HIV-1 Env gp140.</a> J. Virol. 87, 11462-11475 (2013).</li><li>Ringe, R. P., 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=Cleavage+strongly+influences+whether+soluble+HIV-1+envelope+glycoprotein+trimers+adopt+a+native-like+conformation.">Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 18256-18261 (2013).</li><li>Desormeaux, A., 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=The+highly+conserved+layer-3+component+of+the+HIV-1+gp120+inner+domain+is+critical+for+CD4-required+conformational+transitions.">The highly conserved layer-3 component of the HIV-1 gp120 inner domain is critical for CD4-required conformational transitions.</a> J. 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. Virol. 65, 5007-5012 (1991).</li><li>Medjahed, H., Pacheco, B., Desormeaux, A., Sodroski, J., Finzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+gp120+major+variable+regions+modulate+cold+inactivation.">The HIV-1 gp120 major variable regions modulate cold inactivation.</a> J. Virol. 87, 4103-4111 (2013).</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=Characterization+of+conserved+human+immunodeficiency+virus+type+1+gp120+neutralization+epitopes+exposed+upon+gp120-CD4+binding.">Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding.</a> J. Virol. 67, 3978-3988 (1993).</li><li>Finzi, A., 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=Topological+layers+in+the+HIV-1+gp120+inner+domain+regulate+gp41+interaction+and+CD4-triggered+conformational+transitions.">Topological layers in the HIV-1 gp120 inner domain regulate gp41 interaction and CD4-triggered conformational transitions.</a> Mol. Cell. 37, 656-667 (2010).</li><li>Kassa, A., 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=Transitions+to+and+from+the+CD4-bound+conformation+are+modulated+by+a+single-residue+change+in+the+human+immunodeficiency+virus+type+1+gp120+inner+domain.">Transitions to and from the CD4-bound conformation are modulated by a single-residue change in the human immunodeficiency virus type 1 gp120 inner domain.</a> J. Virol. 83, 8364-8378 (2009).</li><li>Xiang, S. 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=Mutagenic+stabilization+and/or+disruption+of+a+CD4-bound+state+reveals+distinct+conformations+of+the+human+immunodeficiency+virus+type+1+gp120+envelope+glycoprotein.">Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein.</a> J. Virol. 76, 9888-9899 (2002).</li><li>Mouquet, 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=Complex-type+N-glycan+recognition+by+potent+broadly+neutralizing+HIV+antibodies.">Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 3268-3277 (2012).</li><li>Julien, J. P., 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=Broadly+neutralizing+antibody+PGT121+allosterically+modulates+CD4+binding+via+recognition+of+the+HIV-1+gp120+V3+base+and+multiple+surrounding+glycans.">Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.</a> PLoS Pathogens. 9 (e1003342), (2013).</li><li>Walker, L. 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=Broad+neutralization+coverage+of+HIV+by+multiple+highly+potent+antibodies.">Broad neutralization coverage of HIV by multiple highly potent antibodies.</a> Nature. 477, 466-470 (2011).</li><li>Moore, J. P., Willey, R. L., Lewis, G. K., Robinson, J., 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=Immunological+evidence+for+interactions+between+the+first,+second,+and+fifth+conserved+domains+of+the+gp120+surface+glycoprotein+of+human+immunodeficiency+virus+type+1.">Immunological evidence for interactions between the first, second, and fifth conserved domains of the gp120 surface glycoprotein of human immunodeficiency virus type 1.</a> J. Virol. 68, 6836-6847 (1994).</li><li>Robinson, J. E., Yoshiyama, H., Holton, D., Elliott, S., Ho, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+antigenic+sites+on+HIV+gp120+identified+by+a+panel+of+human+monoclonal+antibodies.">Distinct antigenic sites on HIV gp120 identified by a panel of human monoclonal antibodies.</a> J. Cell. Biochem. Suppl. 16E (Q449), (1992).</li><li>Bonsignori, 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=Antibody-Dependent+Cellular+Cytotoxicity-Mediating+Antibodies+from+an+HIV-1+Vaccine+Efficacy+Trial+Target+Multiple+Epitopes+and+Preferentially+Use+the+VH1+Gene+Family.">Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family.</a> J. Virol. 86, 11521-11532 (2012).</li><li>Brand, D., Srinivasan, K., 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=Determinants+of+human+immunodeficiency+virus+type+1+entry+in+the+CDR2+loop+of+the+CD4+glycoprotein.">Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein.</a> J. Virol. 69, 166-171 (1995).</li></ol>

The authors declare no conflicts of interest.&#160;

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 implies that it is one of its most important pitfalls. First off, antigens to be studied with this technique are required to be available in an independent expression vector. As such, Env genes from various clinical sources or proviral constructs would need to be subcloned into mammalian expression vectors. While full-length proviral constructs can also be used in this technique (see Veillette&#160;et al.18), this also implies active viral particles production thus requiring work in appropriate biocontainment facilities.
Moreover, success of this technique is intimately linked with transfection efficiency. Low signals obtained are often due to poor expression of transfected antigens. Typical sources of problems are plasmid DNA quality, transfection reagents. and cells viability. If required, optimization of transfection conditions could also be performed using other techniques, such as flow cytometry or western blotting. Of note, it is also important to be aware that expression of some Env constructs could be suboptimal and could therefore affect the technique&#8217;s outcome.
Here we focused on probing HIV-1 Env conformation using previously described CD4i mAbs. This setting allows for a broad range of analysis such as probing the effect of Env point mutations or the conformational consequences of coexpressed proteins. Moreover, the technique described here can also be used with well-characterized Env mutants with different conformational propensity in order to probe the specificity of different mAbs for various Env conformations. This allows an easy and rapid characterization of newly isolated mAbs while not requiring highly-specialized equipment or know-how.
Although we only used this method against Env from various HIV-1 clades and other close relative lineages (HIV-2, SIV/Mac)18, we believe this assay could be adapted for additional surface antigens, such as ones from other virus families.

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><tbody><tr><td>HOS cells</td><td>ATCC</td><td>CRL-1543</td><td></td></tr><tr><td>White Opaque Tissue Culture Plate, 96-well, Flat Bottom</td><td>BD</td><td>353296</td><td></td></tr><tr><td>Polyethylenimine, linear, 25,000 MW</td><td>Polysciences</td><td>23966</td><td>Prepared in 1 mg/ml solution</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11995</td><td></td></tr><tr><td>Goat Anti-human IgG, Peroxidase Conjugated</td><td>Pierce</td><td>31413</td><td></td></tr><tr><td>Enhanced Chemiluminescence Substrate</td><td>PerkinElmer</td><td>NEL105001EA</td><td></td></tr><tr><td>TriStar LB 941, Plate Reader</td><td>Berthold Technologies</td><td></td><td></td></tr></tbody></table>

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 that expose CD4i mAbs 17b and 48d epitopes which overlap the coreceptor binding site24,29, whereas the outer-domain recognizing mAb 2G12 is not affected by this treatment&#160;as expected18,24.
The impact of point mutations in Env conformation can also be assessed using this assay, as presented in Figure 3. Here we used either the layer 1 Env mutant H66A, known to have a decreased propensity to spontaneously sample the CD4-bound conformation18,24,30,31 or a mutant (S375W) which predisposes Env to the CD4-bound state32 and obtained concordant results (Figure 3A). In cases where different Env expressors are used, it is often necessary to normalize the raw data expressed as relative light units (RLU) according to expression levels. In this case, we used PGT121, a mAb recognizing part of the Env glycan shield33-35, as the normalizing antibody (Figure 3B).
As we recently described18, interaction of Env and CD4 in the same cell leads to Env conformational changes that expose CD4i epitopes. In Figure 4, we cotransfected increasing amounts of a CD4 expressor together with Env in the cell-based ELISA assay and obtained increasing signals for CD4i mAbs A32 and C1118,36-38, which recognize discontinuous epitopes in the inner domain of gp120, whereas Env recognition by the conformational-independent 2G12 antibody was not affected (Figure 4A). In order to control for transfection efficiency between conditions, raw data was normalized to 2G12 (Figure 4B). Increased signals obtained for A32 and C11 antibodies depended on Env-CD4 interaction as indicated by the absence of A32 and C11 modulation when Env was cotransfected with a CD4 mutant (F43H) with decreased ability to interact with Env39.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig1highres.jpg" width="500" /> 
Figure 1.&#160;Schematic representation of the anti-Env cell-based ELISA.&#160;(A) General scheme of the procedure in which HOS cells are transfected to express trimeric Env at the cell surface. Env conformation can then be sampled by using different antibodies recognizing specific conformations (such as CD4i mAbs). Signals are detected by chemiluminescence after staining with HRP-conjugated anti-human mAbs. sCD4 (B) or coexpression of cellular CD4 (C) can be used to induce Env conformational changes that lead to exposure of CD4i epitopes.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig2highres.jpg" width="500" /> 
Figure 2.&#160;sCD4 induces Env conformational changes leading to exposure of CD4i epitopes. Interaction of sCD4 with HIV-1CO-JRFL&#916;CT Env enhances recognition by antibodies targeting CD4i epitopes (17b, 48d), but not by the gp120 outer-domain recognizing antibody 2G12. A plasmid encoding HIV-1CO-JRFL&#916;CT Env was transfected in each well and 48 hr later the cells were washed and incubated in presence or absence of 4 &#181;g/ml sCD4 for 30 min at RT before continuing with the standard protocol (Day 4 &#8211; ELISA). Env conformation was then probed by incubating with 0.25 &#181;g/ml 2G12, 1 &#181;g/ml 17b or 48d anti-Env mAbs for 1 hr at RT. Signals were detected by chemiluminescence after incubation with an HRP-conjugated anti-human antibody for 45 min at RT. Shown are the mean RLU values &#177; SD of six replicates with signal obtained from wells transfected with an irrelevant plasmid (no Env) subtracted. Data is representative of results obtained in three independent experiments, with significance tested by two-way ANOVA (ns, not significant; ****, p &#60;0.0001).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig3highres.jpg" width="500" /> 
Figure 3.&#160;Modulation of Env conformation.&#160;HIV-1 YU2&#916;CT layer 1 Env mutant (H66A) diminishes CD4i 17b recognition whereas the S375W variant exhibits increased 17b signal and is sufficient to restore the phenotype of the layer 1 mutant. (A) RLU values of the signals obtained using anti-Env PGT121 and 17b mAbs. (B) PGT121-normalized signals of CD4i mAb 17b following treatment with or without sCD4. Shown are the mean values &#177; SD of triplicates with signal obtained from wells transfected with an irrelevant plasmid (no Env) subtracted. Data is representative of results obtained in three independent experiments, with significance tested by two-way ANOVA (**, p &#60;0.01; ***, p &#60;0.001; ****, p &#60;0.0001).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig4highres.jpg" width="500" /> 
Figure 4.&#160;Coexpression of cellular CD4 enhances recognition by CD4i antibodies.&#160;A CD4-encoding plasmid was cotransfected with HIV1YU2&#916;CT Env in order to favor the CD4-bound conformation18. (A) RLU values of the signals obtained using anti-Env 2G12, A32 or C11 mAbs and the anti-CD4 OkT4 mAb. (B) 2G12-normalized signals of CD4i mAbs A32 and C11. Shown are the mean values &#177; SD of triplicates with signal obtained from wells transfected with an irrelevant plasmid (no Env) subtracted. Data is representative of results obtained in three independent experiments. Grey bar indicates in absence of CD4, whereas the increasing blue bar indicates a step-wise increase in the amount of CD4 expressor being transfected (1.7 ng, 3.5 ng, and 7 ng) and red bar indicates the transfection of a CD4 mutant (F43H, 7 ng) with decreased capacity to interact with gp120.

Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

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 ...

conformational-evaluation-hiv-1-trimeric-envelope-glycoproteins-using

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.3K Views.  Université de Montréal. 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.

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

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' ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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 ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

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

Summary

Explore More Videos

Chapters in this video

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

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

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy