Name: conformational evaluation of hiv-1 trimeric envelope glycoproteins using a cell-based elisa assay
Uploaded: 2014-09-14T15:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay at JoVE.com

我们感谢詹姆斯·罗宾逊博士的慷慨的A32，17B，48D的礼物，和C11单克隆抗体。 PGT 121通过美国国立卫生研究院艾滋病试剂项目，艾滋病，NIAID，美国国立卫生研究院（目录号12343）中科获得。这项工作是由加拿大创新基金会项目负责人＃29866的支持，由CIHR的操作＃257792，由FRQS设立的青年科学家给予＃24639到自动对焦，并通过CRCHUM连续补助金以及由CIHR资助的催化剂＃126630房颤和MR。房颤是一种FRSQ Chercheur布西尔少年团契1＃24639的收件人。 MV由CIHR博士后研究奖＃291485支持。

1日1 - 细胞培养<ol><li>板为2×10 4人骨肉瘤（HOS）在一个不透明的，96孔细胞培养适于发光读板，每孔的细胞。使用的Dulbecco改良的Eagle培养基（DMEM），补充有10％胎牛血清（FBS）和100 U / ml的青霉素 - 链霉素。孵育直到第二天，在37℃，5％CO 2。 </li></ol> 2，第2天 - 聚乙烯亚胺（PEI）转<ol><li>根据后续步骤准备转染混合物。根据井是用相同的信封被转染的数量调节的试剂和DNA的量。 </li><li>管答:加入10纳克达编码质粒（如PTAT-Ⅲ27）和150纳克ENV-质粒编码为5微升补充有25毫米肝素钠。 </li><li>康达编码的质粒是用达依赖性包膜编码质粒如pSVIII时才是必需的。 </li><li> B管:加入450纳克PEI（从1微克/微升的溶液），以5微升DMEM中。 </li><li>加入B管的内容到管A.充分混合，通过涡旋10秒，孵育转染混合物在室温（22℃）10分钟。 </li><li>加10微升每96孔板的转染混合物中。孵育48小时，在37℃，5％CO 2。 </li></ol> 3，第4天 - 酶联免疫吸附<ol><li>执行所有的实验在室温下，以尽量减少包膜/抗体复合物可能的内吞作用。 </li><li>制备250毫升每板清洗缓冲液被用于在同一时间。洗涤缓冲液为1x Tris缓冲盐水（TBS）pH为7.5（50毫摩尔Tris-CL，pH值7.5; 150mM的NaCl）中，补充有1毫米MgCl 2和1.8mM的氯化钙 。 </li><li>通过加入1％脱脂奶粉和5mM的Tris pH值8.0至洗涤缓冲液制备125毫升每板封闭缓冲液。 </li><li>从96孔板中除去细胞培养基和转染混合物（上清液）。 </li><li>加入100μ，L封闭液，每孔孵育20分钟在室温中。 </li><li>除去上清液，加入50微升每孔抗体（或血清）中，稀释至适当浓度的封闭液。通常情况下，使用1微克/毫升的浓度。孵育1小时，在室温下。 </li><li>洗3次用100微升封闭液，然后重复洗涤过程3次用100微升洗涤缓冲液。 </li><li>取出上清液，加入100μl封闭液，孵育5分钟在室温。 </li><li>除去上清液，加入50微升二抗，在封闭缓冲液稀释1/3000。根据制造商的不同而变化的最佳抗体稀释。孵育40分钟，在室温。 </li><li>洗3次用100微升封闭液，然后重复洗涤过程3次用100微升洗涤缓冲液。 </li></ol> 4，数据采集<ol><li>从板除去上清液，每孔加入30μl的1个增强的化学发光（ECL）的衬底。 </li><li>获得化学1秒iluminescence信号/孔上，根据制造商的说明合适的平板阅读器。读取时间可根据硬件上的差异不同。 </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>

作者宣称没有利益冲突。

该测定被优化以检测特异性单克隆抗体的相互作用与HIV-1三聚体包膜表达在细胞表面。一旦该协议已经建立，它可用于在中到高吞吐量和低的总材料成本和抗体的小的量。由于该测定是染为基础，它可以很容易地以研究它们对包膜构象效应适于胞内蛋白如CD4的共表达。 然而，该协议的转基也意味着，这是其最重要的缺陷之一。首先，抗原被研究这种技术是必需的，以提供在一个独立的表达载体中。因此，从各种临床来源的或原病毒构建体env基因需要被亚克隆到哺乳动物表达载体中。而全长前病毒构建体也可在此技术中使用（参见维耶特等人 18），这也意味着作为香港专业教育学院的病毒颗粒的生产，因此需要在适当的生物防护设施的工作。 此外，该技术的成功是密切与转染效率联系在一起的。得到的低信号往往是由于转染抗原的表达差。典型的问题来源是质粒DNA的质量，转染试剂。和细胞活力。如果需要的话，也可以使用其它技术，如流式细胞术或免疫印迹中进行的转染条件的优化。值得注意的是，它也是重要的是要注意，一些包膜构建体的表达可能是不理想的，因此，可能会影响到该技术的结果。 在这里，我们着重于探讨利用前面所述的CD4i单克隆抗体的HIV-1包膜构象。此设置允许各种分析，如探测的信封点突变或共表达的蛋白质的构象后果的影响。此外，该技术描述了他重也可与充分表征的信封的突变体具有不同的构象的倾向，为了探讨不同的mAbs用于各种包膜构象的特异性使用。这使得新分离的单克隆抗体，而不是需要高度专业化的设备或技术诀窍一种简单，快速的特性。 虽然我们只用这种方法对信封从不同的HIV-1的分支及其他近亲属谱系（HIV-2，SIV /苹果机）18，我们认为此法可以适用于更多表面抗原，如其他病毒家族的人。

人类免疫缺陷病毒1型（HIV-1）的条目，以三聚体的病毒包膜糖蛋白（环境）介导的感染周期的第一步骤。是唯一外露的病毒抗原，在病毒粒子的表面呈现，将信封三聚体引发中和非中的抗体。因此，它表示用于疫苗的免疫原设计了一个有趣的候选者。然而，信封疫苗试验中的可溶性或重组的形式引起的反应，只有对最初级的HIV-1分离株1-3最小的有效性。尽管如此，部分疗效的HIV-1包膜的RV144疫苗试验4新的兴趣作为免疫原的候选观察。这是证实了最近的一项研究说明了疫苗引起的抗包膜抗体均足以产生一定程度的保护，防止SIV和HIV挑战5。 后所要合成的内质网中，包膜糖蛋白在前驱物的gp160，经历，这对于其对燃料的病毒融合过程能力的关键不同的翻译后修饰。被裂解成其特细胞质gp120和gp41的跨膜亚基6-10，与非共价相互作用维持的gp120-gp41的联系之前，信封前体必须正确和准倍三聚体。受感染的细胞机器还负责大量糖基化的包膜，其包含约50％的总质量为11,12。所得到的复合结构允许信封是构象柔性13,14，同时提供了被认为是允许信封，以适应和隐藏某些高度免疫原性表位，否则将暴露15-19亚稳态，突出的重要性，以更好地理解不同的构象由天然包膜三聚体取样。 迄今为止，一些技术已经开发并成功地用于研究包膜构象沟道安格斯。然而，他们在他们的局限性会发生变化，而通常被限制在特定的包膜上下文。例如，表面等离子体共振，或使用构象特异性单克隆抗体的免疫沉淀实验（单抗），依赖于任一的单体的水溶性或可溶的信封分子是已知的，从它们的三聚体形式的20,21 immunogenetically不同。最近的研究还表明，裂解影响导致主要抗原决定簇所认可非中抗体14,22,23曝光信封构象。 在这里，我们详细描述了一种方法，它允许快速和容易的决心cellularly表达信封三聚体18,24-26的构象。下列信封的人贴壁细胞系的瞬时转染包膜特异性抗体的结合使用了一个简单的化学发光反应进行检测。这种技术也可以用来表征的构象偏好构象依赖新生凹痕抗体。因此，这个实验提供了一个强大且高度灵活的检测方法。

<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.

使用上述的一般方法中，我们适应的协议，以测定在野生型（WT）或突变env可溶性CD4（SCD4）和共表达细胞的CD4上CD4i表位的暴露的影响，如前所述18,24， 25,28:图1示意性地表示的一般方法和CD4i表位的下列与SCD4或通过蜂窝CD4 18的共表达治疗的曝光。在图2中，我们使用SCD4诱导的暴露CD4i单抗17B和48D的表位重叠的共受体的结合位点24,29信封的构象变化，而 ​​所述外结构域识别的mAb 2G12不会受到这种治疗预期18,24。 在信封的构象点突变的影响，也可以用此试验评估，如在图3中给出，在这里我们使用，也可以将第1层包膜突变H66A，已知具有降低ð倾向自发采样的CD4结合的构象18,24,30,31或突变（S375W），该预先处理信封的CD4结合状态32，并获得一致的结果（ 图3A）。在不同的包膜表达者使用的情况下，它通常是必要的，以归一化表示为根据表达水平的相对光单位（RLU）的原始数据。在这种情况下，我们使用PGT121的包膜糖屏蔽33-35的单克隆抗体识别的部分，作为归一化的抗体（ 图3B）。 正如我们最近描述18，信封和CD4在同一细胞相互作用导致ENV，揭露CD4i表位的构象变化。在图4中 ，我们共转染增加CD4细胞膨的量连同信封中的基于细胞的ELISA测定法和获得CD4i的mAb A32和C11 18,36-38，其识别不连续的表位中GP12的内域增加信号0，而信封识别的构象独立的2G12抗体并没有受到影响（ 图4A）。为了控制为条件之间的转染效率，原始数据进行归一化，以2G12（ 图4B）。获得A32增加了信号和C11抗体依赖ENV-CD4相互作用所没有的A32和C11调制所示，当信封被转染与CD4细胞突变（F43H）有能力的下降与信封39交互。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig1highres.jpg" width="500" /> 其中HOS细胞被转染以表达三聚体包膜在细胞表面的过程的抗ENV基于细胞的ELISA（A）的总方案的图1的示意图 。被膜构象可以然后通过使用识别特定的不同抗体进行采样构（如CD4i单抗）。信号是通过化学发光与HRP偶联的抗人单克隆抗体染色后进行检测。 SCD4（B）或细胞的CD4（C）的共表达可用于诱导导致CD4i表位的暴露包膜构象变化。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig2highres.jpg" width="500" /> 图2 SCD4诱导信封导致CD4i表位 SCD4用。相互作用的暴露构象改变的HIV-1的CO-JRFLΔCT信封增强识别通过靶向CD4i表位（图17B，48D）的抗体，而不是由gp120的外结构域识别抗体2G12。一种质粒编码HIV-1的CO-JRFLΔCT信封转染在各孔中，48小时后，将细胞在室温下洗涤和孵育在存在或不存在的4微克/毫升SCD4 30分钟续前与标准协议inuing（第4天 - ELISA）。被膜构象，然后通过用0.25微克/毫升2G12，1微克/毫升17B或48D抗ENV单克隆抗体孵育1小时，在室温下探测到。孵化与HRP偶联的抗人抗体在室温下45分钟后，通过化学发光法检测信号。显示的是平均RLU值的±从水井转不相关质粒获得了6个重复，信号SD（无信封）中减去。数据是代表在三个独立的实验得到的结果的，具有显着性通过两因素方差分析测试（纳秒，不显著; **** P &lt;0.0001）。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig3highres.jpg" width="500" /> 图3调制信封构象。HIV-1的YU2ΔCT1层包膜突变体（H66A）减少CD4i 17B的认可，而S375W变种展览s提高17b的信号，并且是足以恢复的层1突变体的表型:（A）使用抗-ENV PGT121和17b的单克隆抗体获得的信号的RLU值。 （ 二）CD4i单抗17B以下的有或无SCD4治疗PGT121归一化的信号。显示的是平均值±从水井转不相关质粒获得了一式三份的信号标清（无信封）中减去。数据是代表在三个独立的实验得到的结果的，具有显着性通过两因素方差分析测试（**，P &lt;0.01; ***，P &lt;0.001; **** P &lt;0.0001）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51995/51995fig4highres.jpg" width="500" /> 图4的共表达细胞的CD4由CD4i抗体增强识别。甲CD4编码的质粒共转染HIV1 YU2ΔCT信封以便有利于的CD4结合构象18。 （A）使用抗-ENV 2G12，A32或C11单克隆抗体和抗CD4 OKT4单克隆抗体获得的信号的RLU值。 （B）CD4i的mAb A32和C11的2G12标准化信号。显示的是平均值±从水井转不相关质粒获得了一式三份的信号标清（无信封）中减去。数据代表三个独立实验中获得的结果。灰色条表示在没有的CD4，而越来越多的蓝色条表示的CD4数量的逐步增加膨转染（1.7毫微克，3.5毫微克，而7毫微克）和红条表示CD4细胞突变体转染（F43H 7 NG）与降低产能与gp120的互动。

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的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

14.3K 次观看.  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.

视频: 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.

免疫相关动态三级的HIV - 1 V3环冠R2的序列结构特点的评估从头折叠

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' ...

科研

免疫与感染

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.

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

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.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

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.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

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.

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

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.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

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.

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

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.

体外ELISA测试评估狂犬病疫苗效力

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.

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

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 ...

<meta charset="utf8"></head><body>使用双报告基因和多重技术分析 SARS-CoV-2

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.

<meta charset="utf8"></head><body>作者聚焦:通过新型免疫捕获和质谱技术推进抗病毒策略

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.

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

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