This work was supported by a grant from the German Federal Ministry of Education and Research, funding program Forschung an Fachhochschulen, contract numbers 13FH767IA6 and 13FH242PX6 to JS. Figures 1 and 2 were created with BioRender.com.

1. Sample preparation
<ol>
	<li>Seed the VLP producer suspension cell lines derived from 293-F cells expressing HIV structural genes gag alone or in concert with env 30 at a low cell density of 0.5 x 106 cells per mL in 293-F Expression Medium.</li>
	<li>Let them expand for 3-4 days at 37 &#176;C and 8% CO2 in a shaker incubator rotation with an orbit of 5 cm and 135 rounds per minute (rpm).</li>
	<li>Pellet the producer cells by centrifugation at 100 x g for 5 min. Filter the clarified supernatant to remove residual cells and cell debris using 0.45 &#181;m polyvinylidene fluoride (PVDF) membrane syringe filters to obtain cell-free cell culture supernatant (CFSN). 
	NOTE: The protocol can be paused here. CFSN can be stored overnight at 4 &#176;C.</li>
	<li>Either use the CFSN directly for the experiment or pellet VLPs from 35 mL of CFSN employing ultracentrifugation (112,700 x g, 4 &#176;C, 1.5 h).</li>
	<li>Upon ultracentrifugation, discard the supernatant and suspend the VLP pellets in 200 &#181;L of 15% (w/v) trehalose solution per centrifuge tube. 
	NOTE: The VLP pellet is often not visible to the naked eye. The protocol can be paused here. VLPs can be stored at -80 &#176;C.</li>
	<li>Prior to the VLP capture assay, determine the viral core protein concentrations in the VLP containing samples using an ELISA. This step is important to standardize the capture assay input.</li>
</ol>
2. VLP capture assay
NOTE: An overview of the workflow is depicted in Figure 2.
<ol>
	<li>Suspend the magnetic beads by either pipetting up and down or mixing on a rotator at 50 rpm for at least 5 min.</li>
	<li>Meanwhile, prepare the antibody solution containing the bNAbs. Use 10 &#181;g of each bNAb in 200 &#181;L of antibody binding and washing buffer (see Table of Materials) per reaction. 
	NOTE: Antibody amounts may vary depending on the affinity of the bNAb and antigen decoration density on the VLPs used.</li>
	<li>Use 50 &#181;L of the magnetic bead solution (see Table of Materials) per reaction and transfer the beads into a 1.5 mL reaction tube. Place the tubes on the magnetic separation rack. 
	NOTE: Alternatively, a strong single magnet can be used for each tube to separate the beads from the supernatant.</li>
	<li>Wait a few minutes until the beads gather at the tube wall to ensure that all beads are collected. Remove the supernatant.</li>
	<li>Remove the magnet and suspend the beads in 200 &#181;L of the bNAb solution prepared in step 2.2. Incubate for 30 min to 3 h mixing on a rotator at 50 rpm at room temperature.</li>
	<li>Place the reaction tubes in the magnetic separation rack again, wait and remove the supernatant.</li>
	<li>Remove the tubes from the magnet and wash the beads by resuspending in 200 &#181;L of antibody binding and washing buffer.</li>
	<li>Repeat step 2.4. Remove as much washing buffer&#160;as possible.</li>
	<li>Add the samples to the bead-bound bNAbs. 
	NOTE: The VLP input for the capture assay using HIV-derived particles should be at least 15 ng of viral core protein per reaction. VLP amounts may vary depending on the affinity of the bNAb, and the antigen decoration density on the VLPs used.</li>
	<li>If the sample volume added in step 2.9 is below 1 mL, add PBS to adjust the sample volume to 1 mL. Suspend the beads gently by pipetting.</li>
	<li>Incubate the samples and beads for 2.5 h on a rotator at room temperature. Ensure that the beads stay in suspension and the solution is thoroughly mixed during incubation.</li>
	<li>Place the tubes on the magnet and remove the supernatant.</li>
	<li>Wash the magnetic beads by suspending them in 200 &#181;L of washing buffer (see Table of Materials). Repeat the washing step three times.</li>
	<li>Suspend the beads in 100 &#181;L of washing buffer and transfer the suspension to a clean (heat resistant) reaction tube.</li>
	<li>Place the tube on the magnetic separation rack (see Table of Materials) and remove the supernatant completely.</li>
	<li>Prepare denatured SDS-PAGE samples following steps 2.16.1-2.16.2 or non-denatured samples by eluting the VLPs from the magnetic beads following steps 2.16.3-2.16.5.
	<ol>
		<li>To prepare denatured SDS-PAGE samples, suspend the beads in 20-80 &#181;L of Laemmli buffer (0.8 &#181;L of Laemmli buffer per 1 ng of p55 Gag assay input) and incubate at 95 &#176;C for 5 min.</li>
		<li>Proceed directly with SDS-PAGE or store the samples at -20 &#176;C. 
		CAUTION: Laemmli buffer contains 2-mercaptoethanol and sodium dodecyl sulfate. Wear protective gloves and eye protection. Avoid contact with skin, eyes, and clothes. Do not inhale vapors. Work in a well-ventilated space, e.g.,&#160;a fume hood. 
		NOTE: The protocol can be paused here.</li>
		<li>Alternatively, perform a non-denaturing elution step to obtain VLPs with preserved native protein structure.</li>
		<li>Add 25 &#181;L of elution buffer (frequently provided with the beads) to the magnetic beads and incubate for 2-5 min at room temperature.</li>
		<li>Remove the beads and transfer the eluate to a clean tube. Use the eluate for subsequent assays or store it at 4 &#176;C. 
		&#8203;NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
3. Analysis of captured VLP samples
NOTE: For analysis of the captured VLPs, conduct an SDS-PAGE and subsequently Western blot-analysis31,32.
<ol>
	<li>Place the sample tubes on the magnetic rack to separate the beads from the solution.</li>
	<li>Load 10 &#181;L of each sample into individual wells of the gel. 
	&#8203;NOTE: In this protocol, viral p55 Gag core proteins were detected by employing polyclonal rabbit antibodies directed against HIV Gag and secondary polyclonal chicken anti-rabbit IgG coupled to horseradish peroxidase (HRP) antibodies. Viral core proteins were visualized using chemiluminescence detection.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63137/63137fig02.jpg" /> 
Figure 2: Schematic illustration of key steps of the VLP capture assay using cell-free supernatants. (1) The VLP capture assay starts with the binding of the HIV-1 bNAbs to the protein G-coupled magnetic beads. Meanwhile, the VLP solution with a defined p55 concentration is prepared. The cell-free culture supernatant consists of a mixture of p55 Gag VLPs, host cell proteins, and nucleic acids. (2) The magnetic beads coupled to bNAbs and the VLP solution are transferred into a 1.5 mL reaction tube followed by incubation under rotation. (3) Antigen-displaying VLPs are captured by the bNAbs-coated beads. Separation of these immune complexes from host cell-derived contaminants is performed in a magnetic field. (4) The supernatant is removed by pipetting, and the beads are washed three times to remove unbound VLPs. (5) In the next step, reducing protein loading buffer is added to the immune complexes consisting of magnetic beads coated with bNAbs and captured VLPs. (6) Boiling the sample dissociates bNAbs and target antigen from the magnetic beads and lysis the VLPs. (7) Magnetic beads are separated from the solution in a magnetic field. (8) The protein samples are subjected to SDS-PAGE. (9) Western blot-analysis is performed in order to detect viral p55 Gag core proteins. <a href="https://www.jove.com/files/ftp_upload/63137/63137fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Rold&#227;o, A., Mellado, M. C. M., Castilho, L. R., Carrondo, M. J. T., Alves, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-like+particles+in+vaccine+development.">Virus-like particles in vaccine development.</a> Expert Review of Vaccines. 9 (10), 1149-1176 (2010).</li><li>Noad, R., Roy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-like+particles+as+immunogens.">Virus-like particles as immunogens.</a> Trends in Microbiology. 11 (9), 438-444 (2003).</li><li>Qian, C., 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=Recent+progress+on+the+versatility+of+virus-like+particles.">Recent progress on the versatility of virus-like particles.</a> Vaccines. 8 (1), 139 (2020).</li><li>Zabel, F., K&#252;ndig, T. M., Bachmann, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+humoral+immunity:+On+how+B+cell+responses+are+initiated.">Virus-induced humoral immunity: On how B cell responses are initiated.</a> Current Opinion in Virology. 3 (3), 357-362 (2013).</li><li>Garg, H., Mehmetoglu-Gurbuz, T., Joshi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+Like+Particles+(VLP)+as+multivalent+vaccine+candidate+against+Chikungunya,+Japanese+Encephalitis,+Yellow+Fever+and+Zika+Virus.">Virus Like Particles (VLP) as multivalent vaccine candidate against Chikungunya, Japanese Encephalitis, Yellow Fever and Zika Virus.</a> Scientific Reports. 10 (1), 4017 (2020).</li><li>Hodgins, B., Pillet, S., Landry, N., Ward, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plant-derived+VLP+influenza+vaccine+elicits+a+balanced+immune+response+even+in+very+old+mice+with+co-morbidities.">A plant-derived VLP influenza vaccine elicits a balanced immune response even in very old mice with co-morbidities.</a> PLoS ONE. 14 (1), 0210009 (2019).</li><li>Lai, C. C., 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=Process+development+for+pandemic+influenza+VLP+vaccine+production+using+a+baculovirus+expression+system.">Process development for pandemic influenza VLP vaccine production using a baculovirus expression system.</a> Journal of Biological Engineering. 13, 78 (2019).</li><li>Caldeira, J. C., Perrine, M., Pericle, F., Cavallo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-like+particles+as+an+immunogenic+platform+for+cancer+vaccines.">Virus-like particles as an immunogenic platform for cancer vaccines.</a> Viruses. 12 (5), 488 (2020).</li><li>Nika, 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=An+HER2-displaying+virus-like+particle+vaccine+protects+from+challenge+with+mammary+carcinoma+cells+in+a+mouse+model.">An HER2-displaying virus-like particle vaccine protects from challenge with mammary carcinoma cells in a mouse model.</a> Vaccines. 7 (2), 41 (2019).</li><li>Mohsen, M. O., Zha, L., Cabral-Miranda, G., Bachmann, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+findings+and+recent+advances+in+virus-like+particle+(VLP)-based+vaccines.">Major findings and recent advances in virus-like particle (VLP)-based vaccines.</a> Seminars in Immunology. 34, 123-132 (2017).</li><li>Fontana, D., Garay, E., Cevera, L., Kratje, R., Prieto, C., G&#242;dia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+VLPs+based+on+hiv-1+gag+and+a+fusion+rabies+glycoprotein+induce+specific+antibodies+against+rabies+and+foot-and-mouth+disease+virus.">Chimeric VLPs based on hiv-1 gag and a fusion rabies glycoprotein induce specific antibodies against rabies and foot-and-mouth disease virus.</a> Vaccines. 9 (3), 251 (2021).</li><li>Cervera, 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=Production+of+HIV-1-based+virus-like+particles+for+vaccination:+achievements+and+limits.">Production of HIV-1-based virus-like particles for vaccination: achievements and limits.</a> Applied Microbiology and Biotechnology. 103 (18), 7367-7384 (2019).</li><li>Gonelli, C. A., King, H. A. D., Mackenzie, C., Sonza, S., Center, R. J., Purcell, D. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+HIV-1-based+virus-like+particles+with+increased+incorporation+and+stability+of+membrane-bound+env.">Immunogenicity of HIV-1-based virus-like particles with increased incorporation and stability of membrane-bound env.</a> Vaccines. 9 (3), 1-36 (2021).</li><li>Trkola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+not+as+simple+as+one,+two,+three.">HIV not as simple as one, two, three.</a> Nature. 568, 321-322 (2019).</li><li>Berman, P. 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=Protection+of+chimpanzees+from+infection+by+HIV-1+after+vaccination+with+recombinant+glycoprotein+gp120+but+not+gp160.">Protection of chimpanzees from infection by HIV-1 after vaccination with recombinant glycoprotein gp120 but not gp160.</a> Nature. 345 (6276), 622-625 (1990).</li><li>Barouch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+development+of+an+HIV-1+vaccine.">Challenges in the development of an HIV-1 vaccine.</a> Nature. 455 (7213), 613-619 (2008).</li><li>DeCaprio, J., Kohl, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation.">Immunoprecipitation.</a> Cold Spring Harbor Protocols. 2020 (11), 449-461 (2020).</li><li>Barret, B., Wood, P. A., Volwiler, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+gamma+globulins+in+human+serum+by+immunoprecipitation.">Quantitation of gamma globulins in human serum by immunoprecipitation.</a> Journal of Laboratory and Clinical Medicine. 55, 605-615 (1960).</li><li>Kaboord, B., Perr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+proteins+and+protein+complexes+by+immunoprecipitation.">Isolation of proteins and protein complexes by immunoprecipitation.</a> Methods in Molecular Biology. 424, 349-364 (2008).</li><li>Lee, S. H., Chu, K. B., Kang, H. J., Quan, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-like+particles+containing+multiple+antigenic+proteins+of+Toxoplasma+gondii+induce+memory+T+cell+and+B+cell+responses.">Virus-like particles containing multiple antigenic proteins of Toxoplasma gondii induce memory T cell and B cell responses.</a> PLoS ONE. 14 (8), 0220865 (2019).</li><li>Lee, Y. T., 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=Intranasal+vaccination+with+M2e5x+virus-like+particles+induces+humoral+and+cellular+immune+responses+conferring+cross-protection+against+heterosubtypic+influenza+viruses.">Intranasal vaccination with M2e5x virus-like particles induces humoral and cellular immune responses conferring cross-protection against heterosubtypic influenza viruses.</a> PLoS ONE. 13 (1), 0190868 (2018).</li><li>Wang, J., 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=Large-scale+manufacture+of+VP2+VLP+vaccine+against+porcine+parvovirus+in+Escherichia+coli+with+high-density+fermentation.">Large-scale manufacture of VP2 VLP vaccine against porcine parvovirus in Escherichia coli with high-density fermentation.</a> Applied Microbiology and Biotechnology. 104 (9), 3847-3857 (2020).</li><li>Swenson, D. 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=Generation+of+Marburg+virus-like+particles+by+co-expression+of+glycoprotein+and+matrix+protein.">Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein.</a> FEMS Immunology and Medical Microbiology. 40 (1), 27-31 (2004).</li><li>Latham, T., Galarza, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+wild-type+and+chimeric+influenza+virus-like+particles+following+simultaneous+expression+of+only+four+structural+proteins.">Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins.</a> Journal of Virology. 75 (13), 6154-6165 (2001).</li><li>Doyle, J., Ray, M., Ouyang, A., Benton, B., Bell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abstract+4877:+High+throughput+proteomic+applications+using+protein+A/G+magnetic+beads.">Abstract 4877: High throughput proteomic applications using protein A/G magnetic beads.</a> Association for Cancer Research (AACR) 102nd Annual Meeting. 20, 4877 (2011).</li><li>Zhu, 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=Electron+tomography+analysis+of+envelope+glycoprotein+trimers+on+HIV+and+simian+immunodeficiency+virus+virions.">Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (26), 15812-15817 (2003).</li><li>Lavado-Garc&#237;a, J., Jorge, I., Boix-Besora, A., V&#225;zquez, J., G&#242;dia, F., Cervera, L. <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+HIV-1+virus-like+particles+and+determination+of+Gag+stoichiometry+for+different+production+platforms.">Characterization of HIV-1 virus-like particles and determination of Gag stoichiometry for different production platforms.</a> Biotechnology and Bioengineering. 118 (7), 2660-2675 (2021).</li><li>Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+current+methods+to+confirm+protein-protein+interactions.">An overview of current methods to confirm protein-protein interactions.</a> Protein &amp; Peptide Letters. 25 (8), 728-733 (2018).</li><li>Rao, V. S., Srinivas, K., Sujini, G. N., Kumar, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+molecular+techniques+to+study+diversity.">Advances in molecular techniques to study diversity.</a> Plant Biotechnology, Volume 1: Principles, Techniques, and Applications. , 341-365 (2017).</li><li>Rosengarten, J. F., Schatz, S., Wolf, T., Barbe, S., Stitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Components+of+a+HIV-1+vaccine+mediate+virus-like+particle+(VLP)-formation+and+display+of+envelope+proteins+exposing+broadly+neutralizing+epitopes.">Components of a HIV-1 vaccine mediate virus-like particle (VLP)-formation and display of envelope proteins exposing broadly neutralizing epitopes.</a> Virology. , 41-48 (2022).</li><li>JoVE, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grundlegende+Methoden+in+der+Zell-+und+Molekularbiologie.+Separating+Protein+with+SDS-PAGE.">Grundlegende Methoden in der Zell- und Molekularbiologie. Separating Protein with SDS-PAGE.</a> JoVE Science Education Database. , (2021).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> Journal of Visualized Experiments: JoVE. (44), e2359 (2010).</li><li>Sheng, S., Kong, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+antigens+and+antibodies+by+immunoaffinity+chromatography.">Separation of antigens and antibodies by immunoaffinity chromatography.</a> Pharmaceutical Biology. 50 (8), 1038-1044 (2012).</li><li>Guzzo, C., 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=Virion+incorporation+of+integrin+47+facilitates+HIV-1+infection+and+intestinal+homing.">Virion incorporation of integrin 47 facilitates HIV-1 infection and intestinal homing.</a> Science Immunology. 2 (11), (2017).</li><li>Wei, 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=Bacteria+expressed+hepatitis+E+virus+capsid+proteins+maintain+virion-like+epitopes.">Bacteria expressed hepatitis E virus capsid proteins maintain virion-like epitopes.</a> Vaccine. 32 (24), 2859-2865 (2014).</li><li>Jin, J., Park, C., Cho, S. H., Chung, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+level+of+decoy+epitope+in+PCV2+vaccine+affects+the+neutralizing+activity+of+sera+in+the+immunized+animals.">The level of decoy epitope in PCV2 vaccine affects the neutralizing activity of sera in the immunized animals.</a> Biochemical and Biophysical Research Communications. 496 (3), 846-851 (2018).</li><li>Zhang, X., 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=Lessons+learned+from+successful+human+vaccines:+Delineating+key+epitopes+by+dissecting+the+capsid+proteins.">Lessons learned from successful human vaccines: Delineating key epitopes by dissecting the capsid proteins.</a> Human Vaccines and Immunotherapeutics. 11 (5), 1277-1292 (2015).</li><li>DiGiuseppe, S., Bienkowska-Haba, M., Guion, L. G. M., Keiffer, T. R., Sapp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+papillomavirus+major+capsid+protein+L1+remains+associated+with+the+incoming+viral+genome+throughout+the+entry+process.">Human papillomavirus major capsid protein L1 remains associated with the incoming viral genome throughout the entry process.</a> Journal of Virology. 91 (16), 00537 (2017).</li><li>Wang, J. W., Roden, R. B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L2,+the+minor+capsid+protein+of+papillomavirus.">L2, the minor capsid protein of papillomavirus.</a> Virology. 445 (1-2), 175-186 (2013).</li><li>Binley, 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=Profiling+the+specificity+of+neutralizing+antibodies+in+a+large+panel+of+plasmas+from+patients+chronically+infected+with+human+immunodeficiency+virus+type+1+subtypes+B+and+C.">Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C.</a> Journal of Virology. 82 (23), 11651-11668 (2008).</li><li>Thane, K. E., Davis, A. M., Hoffman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+fluorescent+labeling+and+detection+of+single+extracellular+vesicles+using+nanoparticle+tracking+analysis.">Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis.</a> Scientific Reports. 9 (1), 12295 (2019).</li><li>Mulder, A. 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=Toolbox+for+non-intrusive+structural+and+functional+analysis+of+recombinant+VLP+based+vaccines:+A+case+study+with+hepatitis+B+vaccine.">Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: A case study with hepatitis B vaccine.</a> PLoS ONE. 7 (4), 0033235 (2012).</li></ol>

The authors declare that they have no conflict of interest.

Prior to the VLP capture assay, evaluate the formation of VLPs and the expression of the target antigen in the VLP producer cell lines. Instrumental methods are flow cytometric-analysis of the cell surface expression of the antigen as well as antigen- and viral core protein-specific ELISA of CFSN and pelleted VLPs.
Critical steps of the VLP capture assay are the coating of the beads with capture antibodies - here bNAbs - and the subsequent capture of the antigen-positive VLPs by the antibody-coated beads. Successful coating of the beads with antibodies depends on the choice of the conjugated immunoglobulin (Ig)-binding protein. The donor species as well as the Ig class of the antibodies determine whether protein G- or protein A-conjugated beads are preferable. For most species and Ig classes, protein G is the ligand of choice33. As an alternative to protein A/G-conjugated beads, streptavidin beads for the coating with biotinylated antibodies are available. Beads can also be covalently coupled with antibodies.
The capture of the VLPs by antibody-coated beads depends on thorough mixing, sufficient incubation time, antigen abundance, and affinity of the capture antibody. In our experience, thorough mixing of the antibody-coated beads with the VLP samples is best achieved by the utilization of volumes &#62;500 &#181;L in 1.5 mL tubes under rotation for at least 2 h at room temperature or 4 &#176;C. Another potential hurdle is the too low amount of VLPs in the sample. For antibodies strongly binding the target antigen, VLP inputs as low as 15 ng of Gag protein usually allow for readily detectable amounts of the viral core proteins utilizing Western blot-analysis. However, low-affinity antibodies require higher input amounts, e.g., 100 ng of Gag protein, to obtain conclusive results (Figure 3, bNAb 3).
Some surface antigens are prone to protease degradation. Here, we recommend the addition of protease inhibitors to the VLP samples and incubation at 4 &#176;C. Non-specific adhesion of host cell proteins and VLPs to the bead-bound antibodies is rarely observed and should be excluded by utilizing appropriate negative controls, as we demonstrated here using mock and bald VLP samples and isotype control antibodies. Strategies to reduce non-specific binding include extended washing steps and the addition of casein in the washing buffer34. Furthermore, the capture assay may also be improved by determining the optimal ratio of antibody to antigen-displaying VLP amount.
In the last step of the VLP capture assay, we describe the elution of the immune complexes from the beads by boiling in reducing Laemmli buffer. During this step, the VLPs are disassembled, and the capture antibodies and target antigens are separated from the beads. Notably, the donor species of the primary antibody used in the subsequent Western blot-analysis has to differ from the donor of the capture antibody to avoid the unintended detection of the capture antibody by the secondary anti-donor IgG HRP-conjugated antibodies.
The VLP capture assay presented here provides an easy-to-use and sensitive method to detect neutralization-sensitive epitopes in structural intact target antigens displayed on VLP surfaces. However, the capture assay does not enable direct epitope quantification. ELISA performed with bNAbs are instrumental for this purpose and should be conducted in parallel, particularly if examined VLPs are intended to be used in preclinical studies employing animal models35. This is pivotal, as the amount of antigen can directly correlate with the elicitation of a neutralizing antibody response in immunized animals, as shown for porcine circovirus type 2 (PCV2) vaccines36.
An ideal vaccine should result in the elicitation of bNAbs targeting the neutralization-sensitive&#160;epitopes on the virion surface. The analysis of these epitopes especially referring to their full structural integrity on the particulate vaccine surface is crucial for identifying potential vaccine candidates. This is not only the case for HIV-derived VLPs but also for many other VLP vaccines in development37. Prominent VLP-based vaccines are, for example, derived from non-enveloped or capsid parental viruses such as human papillomavirus (HPV). Unlike HIV-1 particles, which are formed by only one structural core protein, namely p55 Gag, and enveloped by the membrane originating from the VLP producer cell, HPV particles consist of only one or two structural core proteins38,39. Likewise and as presented here for enveloped VLPs, the VLP capture assay may also be applicable to the detection of neutralization-sensitive epitopes of non-enveloped VLPs.
As an alternative to the capture assay, VLP samples can be directly subjected to native PAGE followed by Western blot-analysis using bNAbs and appropriate secondary antibodies coupled to HRP40. However and for the analysis of HIV Env-decorated VLPs, this assay is less sensitive as only a low number of antigen proteins per VLP can be expected. In contrast, the capture assay facilitates the detection of the core proteins abundant at large amounts per VLP, in the case of HIV-derived VLPs more than 3,500 Gag proteins form a VLP27. This allows for the very sensitive indirect detection of epitopes in Env displayed even at low densities on VLPs.
The number of well-established methods to examine neutralization-sensitive&#160;epitopes in surface antigens of VLPs is limited. Labeling the antigens displayed on the VLPs is possible with epitope-specific antibody-fluorophore conjugates and subsequent detection by nanoparticle tracking-analysis (NTA), enabling detection and quantification of VLPs. This method has also been successfully developed and optimized for exosomes presenting cell surface markers41. Also, surface plasmon resonance (SPR) spectroscopy allows for the analysis of interactions between unconjugated neutralizing antibodies and cognate epitopes presented on VLPs. Although not suitable for higher throughput analysis, VLPs can also be labeled with bNAbs coupled to gold particles and subsequent transmission electron microscopic (TEM)-examination42.
In conclusion, the VLP capture assay provides some considerable advantages: (i) Assessment of the structural integrity of neutralization-sensitive&#160;epitopes on the surface of VLPs, (ii) sensitive and indirect detection of antigens even when displayed at low densities on VLPs, and (iii) the method does not require cost-intensive analytical equipment.

Virus-like particles (VLPs) resemble the native virus particle structure while lacking the viral genome, thus providing a high safety profile1,2. VLPs represent an individual class of vaccines increasingly developed due to their high immunogenicity3,4,5,6,7. This is particularly the case for membrane-enveloped VLPs, allowing for the display of not only homologous viral surface antigens but also heterologous antigens such as tumor antigens8,9,10. Figure 1 provides an exemplary overview of the structure of an enveloped antigen-decorated VLP. During the development process of VLP-based vaccines, assays are indispensable enabling the analysis of the respective target antigen displayed on the VLP surface. Such assays should be instrumental to elucidate the composition of a particulate vaccine: (i) Are the VLPs decorated with the respective surface antigen? (ii) Has the surface antigen retained its native structure as demonstrated by epitope recognition of neutralizing antibodies (bNAbs) and (iii) can the structural integrity of the VLPs be confirmed due to the detection of the viral protein mediating VLP formation?
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63137/63137fig01.jpg" /> 
Figure 1: Schematic illustration of a membrane-enveloped VLP. VLPs are formed by immature precursor Gag core proteins and surrounded by a lipid membrane derived from the host cell. The antigens, e.g., envelope glycoproteins, are incorporated into the lipid membrane and displayed on the surface of the VLP (on the right). Antigen-specific antibodies recognize the antigen. On the left, a bald VLP without antigen decoration is shown. <a href="https://www.jove.com/files/ftp_upload/63137/63137fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Especially VLPs formed by the viral group-specific antigen (Gag) core precursor protein p55 of human immunodeficiency virus type 1 (HIV-1) are preferred scaffolds for antigen display in vaccine development as numerous antibodies, and ELISA kits are available, enabling the quantification of these VLPs11,12. The HIV-1 envelope glycoproteins (Env), namely, the transmembrane protein gp41 (gp41-TM) and the soluble surface unit gp120 (gp120-SU) forming heterodimers, are incorporated into the membrane envelope of particles and are crucial target antigens for the development of vaccines against HIV infection13,14,15. The display of neutralization-sensitive epitopes in these target antigens is a prerequisite for eliciting a broadly neutralizing antibody response in vaccinees. Besides a T cell response directed against the Gag proteins, this is considered an important correlate of protection against HIV infection16. Consequently, and upon design and production of VLPs decorated with target antigen candidates, the subsequent analysis of the quality of the displayed antigens represents a critical step in the process of vaccine development.
Immunoprecipitation (IP) is a widely used technique for the detection of protein-protein interactions and the purification of protein complexes at a small scale17. Barret et al. first reported on the development of IP in 1960, yet, this method has been constantly further improved. IP enables the capture and isolation of a target antigen (prey) from a solution by employing an antigen-specific antibody (bait) immobilized by coupling to beads18,19. In this protocol, we demonstrate a variation of the classical IP application using membrane-enveloped p55 Gag-formed VLPs as prey and bNAbs that recognize neutralization-sensitive epitopes in the envelope proteins displayed on the surface of the VLPs as bait proteins. The successful application of this VLP capture assay facilitates the prediction of whether the tested antigen-positive VLPs will be able to elicit a neutralizing B cell response in vaccinated people. Such immunogenic properties of VLP-based vaccine candidates are frequently demonstrated in small animal models20,21,22.
In order to assess the quality of the newly developed VLP vaccine candidate, VLP capture assays have been successfully used5,23,24. However, the number of published methods is limited. The VLP capture assay presented here starts with the immobilization of Env-specific bNAbs on protein G-conjugated beads, which bind to the Fc region of mammalian-derived antibodies. Typical matrices for the immobilization of the antibody of choice are agarose or magnetic beads. However, magnetic beads are favorable for high-throughput applications25. In the next step, VLPs displaying the target antigen are captured by bNAb-coated beads. The formed immune complexes consisting of Env-positive VLPs and immobilized bNAbs are easily enriched using a magnet. The isolated immune complexes are eluted in the final step. Subsequently, the VLPs can be biochemically characterized. Here, we performed Western blot-analysis employing p55 Gag viral core protein-specific antibodies to demonstrate that the precipitated target Env antigens were not only harboring the neutralization-sensitive epitopes but were also displayed on Gag-formed VLPs. Furthermore, the detection of the viral core Gag proteins increases the sensitivity of the capture assay since Gag proteins are more abundant than Env in a VLP. In HIV-1, Env proteins are only present at a single- or double-digit number26, whereas more than 3,500 Gag molecules form the core of a particle27.
Compared to other techniques for the examination of protein-protein interactions28,29, the VLP capture assay provides an alternative method for research laboratories not having access to expensive analytical instruments. For example, transmission electron microscopic-analysis (TEM), surface plasmon resonance spectroscopy (SPR), and nanoparticle tracking-analysis (NTA) can be cost-intensive. The capture assay presented here also allows later subjection of captured antigen-positive VLP samples to further protein characterization, e.g., employing gel electrophoresis, immunoblotting, electron microscopy, and mass spectrometry (MS), respectively. Considering that the native structure of the target antigen is preserved during the VLP capture assay, also the performance of a native PAGE and subsequent immunoblotting techniques can be utilized.
The VLP capture assay represents an easy-to-use and sensitive method to examine the decoration of VLPs with target antigens exposing neutralization-sensitive&#160;epitopes, and thus their utility as future vaccine candidates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL reaction tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>4%&#8211;15% Mini-PROTEAN TGX stain-free protein gels</td>
			<td>BioRad</td>
			<td>4568085</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies (bnAbs)</td>
			<td>Polymun Scientic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype control antibody</td>
			<td>invitrogen (Thermo Fisher Scientific)</td>
			<td>02-7102</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc XRS+ imaging system</td>
			<td>BioRad</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-rabbit IgG HRP-coupled</td>
			<td>Life technologies</td>
			<td>A15987</td>
			<td>1:5000 in TBS-T + 2 % (w/v) powdered milk</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G Immunoprecipitation Kit</td>
			<td>invitrogen (Thermo Fisher Scientific)</td>
			<td>10007D</td>
			<td>includes buffers and washing solutions</td>
		</tr>
		<tr>
			<td>FreeStyle 293-F cells</td>
			<td>invitrogen (Thermo Fisher Scientific)</td>
			<td>R790-07</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeStyle 293 Expression Medium</td>
			<td>invitrogen (Thermo Fisher Scientific)</td>
			<td>12338026</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel blotting papers</td>
			<td>Whatman</td>
			<td>GB005</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>0079</td>
			<td>blotting buffer</td>
		</tr>
		<tr>
			<td>Magnetic separation rack</td>
			<td>New England Biolabs</td>
			<td>S1509S</td>
			<td>for 12 x 1.5 mL or 6 x 1.5 mL tubes</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>4627</td>
			<td>blotting buffer</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell electrophoresis system</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler prestained protein ladder</td>
			<td>Thermo Scientific</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylidene fluoride (PVDF) syringe filters, 0.45 &#181;m</td>
			<td>Carl Roth</td>
			<td>KC89.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Powdered milk</td>
			<td>Carl Roth</td>
			<td>T145</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>PVDF transfermembrane, 0.45 &#181;m</td>
			<td>Carl Roth</td>
			<td>T830.1</td>
			<td></td>
		</tr>
		<tr>
			<td>QuickTiter HIV p24 ELISA</td>
			<td>Cell Biolabs</td>
			<td>VPK-108-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal to HIV1 p55 + p24 + p17</td>
			<td>abcam</td>
			<td>ab63917</td>
			<td>1:2000 in TBS-T + 2 % (w/v) powdered milk</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Heidolph</td>
			<td>REAX2</td>
			<td></td>
		</tr>
		<tr>
			<td>ROTI Load 1 (laemmli buffer)</td>
			<td>Carl Roth</td>
			<td>K929.1</td>
			<td>4x concentrated reducing protein gel loading buffer</td>
		</tr>
		<tr>
			<td>ROTIPHORESE 10x SDS-PAGE</td>
			<td>Carl Roth</td>
			<td>3060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>3957</td>
			<td>TBS-T buffer</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34579</td>
			<td></td>
		</tr>
		<tr>
			<td>SW28 rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Cel Media</td>
			<td>basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose dihydrate</td>
			<td>Carl Roth</td>
			<td>8897.2</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Carl Roth</td>
			<td>5429</td>
			<td>blotting buffer</td>
		</tr>
		<tr>
			<td>TRIS hydrochloride</td>
			<td>Carl Roth</td>
			<td>9090</td>
			<td>TBS-T buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Carl Roth</td>
			<td>9127</td>
			<td>TBS-T buffer</td>
		</tr>
		<tr>
			<td>Ultra Clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 3 shows a representative outcome of VLPs first captured from CFSN and VLP pellets, respectively, using bNAbs and subsequently subjected to Western blot-analysis to detect viral core proteins. The beads used for the capture assay were coated with three different bNAbs directed against neutralization-sensitive epitopes of the Env glycoproteins and isotype antibodies serving as negative controls, respectively. Using isotype antibodies-coated beads, no Gag proteins were detectable in samples containing Env-negative (bald VLPs) or Env-displaying VLPs employing Western blot-analysis. This demonstrated that the unspecific binding of VLPs to beads coated with human antibodies did not mediate VLP capture. Bald VLPs were also not bound by bNAbs-coated beads, and consequently, again, no Gag proteins were detectable. In contrast, all three bNAbs captured VLPs displaying Env proteins (Env VLPs), and therefore, Gag proteins were subsequently readily detected. As visible in Figure 3, the capture assay can be utilized to analyze target antigen displaying VLPs in CFSN and pelleted VLP samples, respectively.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63137/63137fig03.jpg" /> 
Figure 3: Detection of neutralization-sensitive&#160;epitopes in HIV-1 envelope glycoproteins displayed on p55 Gag-formed VLPs using broadly neutralizing antibodies. Representative results of Western blot-analysis using viral Gag core protein-specific antibodies after performing a VLP capture assay employing three different broadly neutralizing antibodies (bNAb 1, bNAb 2, and bnAb 3) targeting epitopes within the HIV-1 envelope glycoproteins (Env). Isotype human antibodies pooled from human sera served as negative controls.&#160;Cell culture supernatants were harvested from suspension cell cultures producing VLPs with Env proteins (Env VLPs) and bald VLPs (Env-negative), respectively, as well as from naive cells not expressing any viral proteins (mock). Both the supernatants from the bald VLP producer cell culture as well as from the mock cell culture served as negative controls. Supernatants were freed from contaminating cells using low-speed centrifugation and subsequent filtration to obtain cell-free supernatants (CFSN) for analysis. Upon immunoprecipitation, Western blot-analysis was performed using polyclonal rabbit antibodies directed against Gag and secondary anti-rabbit IgG-HRP conjugates. The apparent molecular weight in kilodaltons (kDa) as visible from the molecular weight marker (MW) is depicted on the left. The arrows indicate the detected precursor protein p55 Gag. (A) For each sample, CFSN containing 100 ng of Gag protein was applied to the capture assay to standardize the input amount of VLPs. CFSN was directly incubated with antibody-coated beads. (B) VLP pellets were obtained by ultracentrifugation of CFSN. Pellets containing 100 ng of Gag protein were applied to the capture assay using isotype antibodies and bNAb 3. Samples for bNAb 1 and bNAb 2 contained only 25 ng of Gag. <a href="https://www.jove.com/files/ftp_upload/63137/63137fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay at JoVE.com

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.

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 ...

detection-neutralization-sensitive-epitopes-antigens-displayed-on

Research

JoVE Journal

Immunology and Infection

3.4K Views.  TH Köln - University of Applied Sciences. 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. 

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

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

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

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...