Name: antigenic liposomes for generation of disease-specific antibodies
Uploaded: 2018-10-25T13:00:00
Description: Watch this Scientific Journal Video about Antigenic Liposomes for Generation of Disease-specific Antibodies at JoVE.com

This research was supported by grants from the Department of Defense (W81XWH-16-1-0302 and W81XWH-16-1-0303).

The general method of coupling protein to lipid and incorporating into liposomes is based largely on earlier work15. All animal procedures described below have been approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee (IACUC). All mice used in the peanut allergy model are BALB/cJ females purchased from at 3 weeks of age. The University of Alberta Animal Care and Use Committee (ACUC) has approved experiments involving use of mouse spleens for e...

<ol>
	<li>Gupta, R. 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=The+prevalence,+severity,+and+distribution+of+childhood+food+allergy+in+the+United+States.">The prevalence, severity, and distribution of childhood food allergy in the United States.</a> Pediatrics. 128 (1), e9-e17 (2011).</li><li>Sicherer, S. H., Munoz-Furlong, A., Godbold, J. H., Sampson, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=US+prevalence+of+self-reported+peanut,+tree+nut,+and+sesame+allergy:+11-year+follow-up.">US prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year follow-up.</a> Journal of Allergy and Clinical Immunology. 125 (6), 1322-1326 (2010).</li><li>Kim, E. 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=Sublingual+immunotherapy+for+peanut+allergy:+clinical+and+immunologic+evidence+of+desensitization.">Sublingual immunotherapy for peanut allergy: clinical and immunologic evidence of desensitization.</a> Journal of Allergy Clinical Immunology. 127 (3), 640-646 (2011).</li><li>Vickery, B. 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=Sustained+unresponsiveness+to+peanut+in+subjects+who+have+completed+peanut+oral+immunotherapy.">Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy.</a> Journal of Allergy and Clinical Immunology. 133 (2), 468 (2014).</li><li>Jones, S. 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=Epicutaneous+immunotherapy+for+the+treatment+of+peanut+allergy+in+children+and+young+adults.">Epicutaneous immunotherapy for the treatment of peanut allergy in children and young adults.</a> Journal of Allergy and Clinical Immunology. 139 (4), 1242 (2017).</li><li>Varshney, 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=A+randomized+controlled+study+of+peanut+oral+immunotherapy:+clinical+desensitization+and+modulation+of+the+allergic+response.">A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response.</a> Journal of Allergy Clinical Immunology. 127 (3), 654-660 (2011).</li><li>Anagnostou, 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=Assessing+the+efficacy+of+oral+immunotherapy+for+the+desensitisation+of+peanut+allergy+in+children+(STOP+II):+a+phase+2+randomised+controlled+trial.">Assessing the efficacy of oral immunotherapy for the desensitisation of peanut allergy in children (STOP II): a phase 2 randomised controlled trial.</a> Lancet. 383 (9925), 1297-1304 (2014).</li><li>Sampson, H. 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=Effect+of+Varying+Doses+of+Epicutaneous+Immunotherapy+vs+Placebo+on+Reaction+to+Peanut+Protein+Exposure+Among+Patients+With+Peanut+Sensitivity:+A+Randomized+Clinical+Trial.">Effect of Varying Doses of Epicutaneous Immunotherapy vs Placebo on Reaction to Peanut Protein Exposure Among Patients With Peanut Sensitivity: A Randomized Clinical Trial.</a> The Journal of the American Medical Association. 318 (18), 1798-1809 (2017).</li><li>Bednar, K. 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=Human+CD22+Inhibits+Murine+B+Cell+Receptor+Activation+in+a+Human+CD22+Transgenic+Mouse+Model.">Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.</a> Journal Immunology. 199 (9), 3116-3128 (2017).</li><li>Macauley, M. 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=Antigenic+liposomes+displaying+CD22+ligands+induce+antigen-specific+B+cell+apoptosis.">Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.</a> Journal Clinical Investigation. 123 (7), 3074-3083 (2013).</li><li>Orgel, K. 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=Exploiting+CD22+on+antigen-specific+B+cells+to+prevent+allergy+to+the+major+peanut+allergen+Ara+h+2.">Exploiting CD22 on antigen-specific B cells to prevent allergy to the major peanut allergen Ara h 2.</a> Journal Allergy Clinical Immunology. 139 (1), 366-369 (2017).</li><li>Smarr, C. B., Hsu, C. L., Byrne, A. J., Miller, S. D., Bryce, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-fixed+leukocytes+tolerize+Th2+responses+in+mouse+models+of+allergy.">Antigen-fixed leukocytes tolerize Th2 responses in mouse models of allergy.</a> Journal of Immunology. 187 (10), 5090-5098 (2011).</li><li>Kulis, 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=The+2S+albumin+allergens+of+Arachis+hypogaea,+Ara+h+2+and+Ara+h+6,+are+the+major+elicitors+of+anaphylaxis+and+can+effectively+desensitize+peanut-allergic+mice.">The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.</a> Clinical and Experimental Allergy. 42 (2), 326-336 (2012).</li><li>Dang, T. 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=Increasing+the+accuracy+of+peanut+allergy+diagnosis+by+using+Ara+h+2.">Increasing the accuracy of peanut allergy diagnosis by using Ara h 2.</a> Journal of Allergy Clinical Immunology. 129 (4), 1056-1063 (2012).</li><li>Loughrey, H. C., Choi, L. S., Cullis, P. R., Bally, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedures+for+the+coupling+of+proteins+to+liposomes.">Optimized procedures for the coupling of proteins to liposomes.</a> Journal Immunological Methods. 132 (1), 25-35 (1990).</li><li>Sen, 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=Protein+structure+plays+a+critical+role+in+peanut+allergen+stability+and+may+determine+immunodominant+IgE-binding+epitopes.">Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes.</a> Journal of Immunology. 169 (2), 882-887 (2002).</li><li>Krall, N., da Cruz, F. P., Boutureira, O., Bernardes, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-selective+protein-modification+chemistry+for+basic+biology+and+drug+development.">Site-selective protein-modification chemistry for basic biology and drug development.</a> Nature Chemistry. 8 (2), 103-113 (2016).</li><li>Jimenez-Saiz, 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=Lifelong+memory+responses+perpetuate+humoral+TH2+immunity+and+anaphylaxis+in+food+allergy.">Lifelong memory responses perpetuate humoral TH2 immunity and anaphylaxis in food allergy.</a> Journal Allergy and Clinical Immunology. 140 (6), 1604-1615 (2017).</li><li>Moutsoglou, D. M., Dreskin, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+Treatment+of+Peanut-Allergic+Mice+with+Bortezomib+Significantly+Reduces+Serum+Anti-Peanut+IgE+but+Does+Not+Affect+Allergic+Symptoms.">Prolonged Treatment of Peanut-Allergic Mice with Bortezomib Significantly Reduces Serum Anti-Peanut IgE but Does Not Affect Allergic Symptoms.</a> International Archives of Allergy and Immunology. 170 (4), 257-261 (2016).</li><li>LaMothe, R. 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=Tolerogenic+Nanoparticles+Induce+Antigen-Specific+Regulatory+T+Cells+and+Provide+Therapeutic+Efficacy+and+Transferrable+Tolerance+against+Experimental+Autoimmune+Encephalomyelitis.">Tolerogenic Nanoparticles Induce Antigen-Specific Regulatory T Cells and Provide Therapeutic Efficacy and Transferrable Tolerance against Experimental Autoimmune Encephalomyelitis.</a> Frontiers in Immunology. 9, 281 (2018).</li><li>Srivastava, K. 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=Investigation+of+peanut+oral+immunotherapy+with+CpG/peanut+nanoparticles+in+a+murine+model+of+peanut+allergy.">Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy.</a> J Allergy Clin Immunol. 138 (2), 536-543 (2016).</li><li>Bellinghausen, I., Saloga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+allergic+immune+responses+in+humanized+mice.">Analysis of allergic immune responses in humanized mice.</a> Cellular Immunology. 308, 7-12 (2016).</li><li>Pillai, S., Mattoo, H., Cariappa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+and+autoimmunity.">B cells and autoimmunity.</a> Curr Opin Immunol. 23 (6), 721-731 (2011).</li><li>Mantegazza, R., Cordiglieri, C., Consonni, A., Baggi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+myasthenia+gravis:+utility+and+limitations.">Animal models of myasthenia gravis: utility and limitations.</a> International Journal of General Medicine. 9, 53-64 (2016).</li><li>Berman, P. 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The methods outlined here are a general protocol for the conjugation of a protein to a lipid which enables the display of the protein on liposomal nanoparticles. For very large multi-subunit proteins, this protocol may have limited utility. The ideal method would be the introduction of a site-specific tag that enables a biorthogonal chemical linking strategy to be used. If expressing the protein recombinantly, this can be possible using available site-specific strategies17, and a wide array of fun...

Food allergy affects 8% of children in the United States, and has increased in prevalence over the past decade1. Allergy to peanut affects 1% of children and is not typically outgrown2. Although several promising clinical trials are underway for the treatment of food allergy, including oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epicutaneous immunotherapy (EPIT), there are currently no FDA-approved treatment strategies for desensitizing peanut-allergic individuals3,4,5,6,7,8. Therefore, allergic individuals must strictly avoid allergens to avoid anaphylaxis. Many questions remain regarding routes of sensitization and underlying mechanisms of food allergy development.
Mouse models are a valuable tool for studying the mechanisms of allergy as well as developing new tolerogenic and desensitization therapies9,10,11,12. This is particularly true because the major peanut allergen (Ara h 2; Ah2) in humans is also the dominant allergen in several described mouse models13,14. While mouse models of peanut allergy are invaluable in studying mechanisms of sensitization and tolerance, a drawback is that they can be variable and require the use of adjuvants. More potent immunogens would be one way to minimize the intrinsic variability of such models. Since B-cells are strongly activated by multivalent antigens, antigenic liposomes displaying the allergen are a good option because of their ability to potentially activate B-cells through the B-cell receptor (BCR) while also having the property of efficiently priming the T-cell compartment through being taken up non-specifically by antigen-presenting cells.
Here, we describe a detailed protocol for conjugating protein antigens to liposomal nanoparticles using a facile and modular strategy. Using a surrogate antigen, anti-IgM Fab fragment, we demonstrate how potent such antigenic liposomes can be in stimulating B-cell activation. Antigenic liposomes displaying Ah2 antigen were used to develop a new mouse model of conferred sensitivity. In this model, splenocytes from verified peanut allergic mice, containing peanut-specific memory B- and T-cells, are transferred into na&#239;ve congenic mice. Memory antibody responses are induced by injection of liposomes conjugated with Ah2 into the recipient mice, in order to induce antibodies against Ah2. Followed by only one boost with soluble Ah2, Ah2-specific antibodies give rise to a strong anaphylactic response when these mice are subsequently challenged with Ah2. As mice undergoing the allergic reaction respond in a highly uniform manner and have not received an adjuvant, this approach is a desirable peanut allergy model and the outcomes suggest that it may have utility in other mouse models driven by antigens directed at allergens and possibly autoantigens.

<table>
	<tbody>
		<tr>
			<td>Model 2110 Fraction Collector</td>
			<td>BioRad</td>
			<td>7318122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholestrol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td>Sigma grade 99%</td>
		</tr>
		<tr>
			<td>SPDP</td>
			<td>Thermo Fisher Scientific</td>
			<td>21857</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPC</td>
			<td>Avanti</td>
			<td>850365</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPE-PEG 18:0</td>
			<td>Avanti</td>
			<td>880120</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPE-PEG Maleimide</td>
			<td>Avanti</td>
			<td>880126</td>
			<td></td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Avanti</td>
			<td>610000</td>
			<td>1mL syringe with holder/heating block</td>
		</tr>
		<tr>
			<td>Filters 0.1 &#181;m</td>
			<td>Avanti</td>
			<td>610005</td>
			<td></td>
		</tr>
		<tr>
			<td>Filters 0.8 &#181;m</td>
			<td>Avanti</td>
			<td>610009</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm Filter Supports</td>
			<td>Avanti</td>
			<td>6100014</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Round Bottom Flask</td>
			<td>Sigma</td>
			<td>Z100633</td>
			<td></td>
		</tr>
		<tr>
			<td>Turnover stoppers</td>
			<td>Thermo Fisher Scientific</td>
			<td>P-301398</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Thermo Fisher Scientific</td>
			<td>P-198194</td>
			<td></td>
		</tr>
		<tr>
			<td>Leur Lock</td>
			<td>Thermo Fisher Scientific</td>
			<td>k4201634503</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G50 Beads</td>
			<td>GE Life Sciences</td>
			<td>17004201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G100 Beads</td>
			<td>GE Life Sciences</td>
			<td>17006001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Calf Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>1x RBC lysis Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Indo-1</td>
			<td>Invitrogen</td>
			<td>I1203</td>
			<td></td>
		</tr>
		<tr>
			<td>CD5-PE</td>
			<td>BioLegend</td>
			<td>100608</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-PE-Cy7</td>
			<td>BioLegend</td>
			<td>103222</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170112</td>
			<td>without calcium and magnesium</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fab anti-mouse IgM</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-007-020</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2 anti-mouse IgM</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-006-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Peanut flour</td>
			<td>Golden Peanut Co.</td>
			<td>521271</td>
			<td>12% fat light roast, 50% protein</td>
		</tr>
		<tr>
			<td>Animal feeding needles</td>
			<td>Cadence Science</td>
			<td>7920</td>
			<td>22g x 1.5&#34;, 1.25 mm - straight</td>
		</tr>
		<tr>
			<td>Microprobe thermometer</td>
			<td>Physitemp</td>
			<td>BAT-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal probe for mice</td>
			<td>Physitemp</td>
			<td>Ret-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin, from vibrio cholera</td>
			<td>List Biological Laboratories, Inc.</td>
			<td>100B</td>
			<td>Azide free</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate-bicarbonate buffer</td>
			<td>Sigma</td>
			<td>C3041</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB Stop Solution</td>
			<td>KPL</td>
			<td>50-85-06</td>
			<td></td>
		</tr>
		<tr>
			<td>SureBlue TMB Microwell Peroxidase Substrate</td>
			<td>KPL</td>
			<td>5120-0077</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Immulon 4HBX plate</td>
			<td>Thermo Scientific</td>
			<td>3855</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified soluble Ara h 2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>purified as in: Sen, et al., 2002, Journal of Immunology</td>
		</tr>
		<tr>
			<td>HSA-DNP</td>
			<td>Sigma</td>
			<td>A-6661</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgE anti-DNP</td>
			<td>Accurate Chemical</td>
			<td>BYA60251</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep anti-Mouse IgE</td>
			<td>The Binding Site</td>
			<td>PC284</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Donkey anti-Sheep IgG</td>
			<td>Accurate Chemical</td>
			<td>JNS065003</td>
			<td></td>
		</tr>
		<tr>
			<td>NeutrAvidin Protein, HRP</td>
			<td>ThermoFisher Scientific</td>
			<td>31001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 anti-DNP</td>
			<td>Accurate Chemical</td>
			<td>MADNP105</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat anti-mouse IgG1</td>
			<td>Southern Biotech</td>
			<td>1070-05</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Insulin Syringes</td>
			<td>BD</td>
			<td>329412</td>
			<td>U-100 Insulin, 0.40 mm(27G) x 16.0 mm (5/8&#34;)</td>
		</tr>
		<tr>
			<td>Superfrost Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-14</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>gibco by Life Technologies</td>
			<td>A10492-01</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875093</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td>70 &#956;m Nylon, White, Sterile, Individually packaged</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>10 gels</td>
		</tr>
		<tr>
			<td>NuPAGE LDS buffer, 4X</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>SeeBlue Plus2 Pre-stained standard</td>
			<td>Invitrogen</td>
			<td>LC5925</td>
			<td>500 &#181;L</td>
		</tr>
		<tr>
			<td>NuPAGE MES/SDS running buffer, 20X</td>
			<td>Invitrogen</td>
			<td>NP0002</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>GelCode Blue Stain</td>
			<td>Thermo Scientific</td>
			<td>24590</td>
			<td>500 mL</td>
		</tr>
	</tbody>
</table>

antigenic liposomes for generation of disease-specific antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Conjugation of the protein of interest with DSPE-PEG(2000) can be demonstrated by running a reducing showing an increase in molecular weight compared to the unconjugated protein. Figure 1A shows a representative gel of anti-mouse IgM F(ab) fragment conjugation to PEG-DSPE, which shows a 2&#8211;3 kDa bandshift for the denatured protein. Note that approximately 50% of the protein appears to be modified, which is expected given that 1:1 stoichiometry was achiev...

Watch this Scientific Journal Video about Antigenic Liposomes for Generation of Disease-specific Antibodies at JoVE.com

Antigenic Liposomes for Generation of Disease-specific Antibodies

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

This method can help answer key questions in the field of immunology, such as how are white blood cells activated, and how do allergies to individual allergens develop? The main advantage of this robust and reproducible allergy mouse model is that fewer mice can be used to yield a lower variance with an experimental population. New immunotherapies to control immune responses require robust mouse models as an initial means of testing in vivo efficacy.Thus, the implications of this technique extend toward allergy immunotherapy. Though this method can provide insight into allergies, it can also be applied to other systems, such as autoimmunity in which undesired antibody responses play a key role in disease. Generally, individuals new to this method will struggle because of lack of experience in working with liposomal nanoparticles or an inexperience with the techniques required in the mouse work.Begin by adding 2.5 molar equivalent of the heterobifunctional crosslinker to the protein and placing the reaction on an oscillating shaker at room temperature for approximately one hour. After a one-hour incubation with SPDP, desalt the protein on a column equilibrated in 100-millimolar sodium acetate, and collect the fractions. Determine the 280-nanometer absorbance, and pool the top fractions.Wash the column in PBS, and add 25-millimolar dithiothreitol, or DTT, to the pooled protein fractions for a five to 10-minute incubation. Then, measure the 280 and 343-nanometer absorbances of the protein to allow calculation of the linking ratio based on the molarity of protein and the linker. Next, run the pooled fractions of the PBS-washed column, and collect the fractions for measurement of the 280-nanometer and top fraction pooling as demonstrated.Add the appropriate volume of 100x DSPE-PEG2000-Maleimide to the protein to achieve a 1x, 10-fold molar excess final concentration with gentle swirling. Then, run the reaction overnight under nitrogen in a sealed round-bottom flask. The following day, run the protein over a crosslinked dextran gel bead column, and store the final pooled fractions of lipid-linked protein at four degrees Celsius until their use.Once the correct amount of each lipid has been calculated, combine all of the lipids into a 12-milliliter borosilicate glass test tube, and use a three-milliliter syringe and tubing to carefully blow the chloroform off with nitrogen. Then, lyophilize the solution with 100 microliters of DMSO overnight. The next morning, add one milliliter of lipid-linked protein to the 12-milliliter lipid tube, and sonicate the solution three to four times for 30 to 60 seconds per sonication in a sonication water bath with at least five-minute rests between sonications.After the last sonication, load the sample into one of the extruding syringes placed into the extruder, and place the extruder syringe into the other end of the extruder. The empty syringe will fill as the lipid is extruded through the polycarbonate 0.8-micrometer membrane. Place the fully assembled extruder into a heating block, and gently depress the plunger to empty the syringe.After the last extrusion, transfer the liposomes into a clean vial, and extrude the lipids through polycarbonate 0.2 and 0.1-micrometer membranes as just demonstrated. Then, store the liposomes at four degrees Celsius. To monitor B-cell response to antigenic liposome activation, resuspend 1.5 times 10 to the seventh splenocytes in calcium flux loading buffer, and add 1.5-micromolar Indo-1 to the cells, inverting the solution in the tube several times to mix.After a 30-minute water bath incubation at 37 degrees Celsius, protected from light, add five times the volume of calcium flux loading buffer, and centrifuge the Indo-1-labeled cells. For B-cell gating, stain cells with anti-CD5 and anti-B220 antibody in 0.5 milliliters of loading buffer at four degrees Celsius for 20 minutes, protected from light. At the end of the incubation, wash the cells in fresh calcium flux loading buffer, and resuspend the pellet at one to two times 10 to the seventh cells per milliliter of fresh calcium flux running buffer for storage on ice, protected from light until analysis.Next, add 0.5 milliliters of cells to a capped, five-milliliter, round-bottom, polystyrene tube, and warm the cells to 37 degrees Celsius in a water bath. After three to five minutes, transfer the tube to a 37-degree Celsius water-jacketed chamber connected to a recirculating water bath, and run the tube in the water-jacket on the flow cytometer. After collecting 5, 000 to 10, 000 events per second, allow the cells to stabilize for 15 to 30 seconds, and reinitiate the data acquisition, collecting the data for at least 10 seconds to establish a background reading.At the 10-second mark, quickly remove the tube from the flow cytometer, and add the appropriate experimental concentration of antigenic liposomes. Then, pulse vortex the cells, and read the tube on the cytometer for an additional three to five minutes. To sensitize the animals, deliver 200 microliters of peanut extract containing cholera toxin via oral gavage to each four to five-week-old BALB/c female mouse recipient once a week for three weeks and 300 microliters of diluted peanut extract in the fourth week.On day 28, prepare peanut extract to a final concentration of one milligram per milliliter in PBS, and use a rectal thermometer to measure the baseline body temperature of each sensitized animal. When all of the temperatures have been measured, administer 200 microliters of peanut extract to each recipient via intraperitoneal injection, and measure the body temperature with the rectal thermometer every 15 minutes for one hour after injection. A dip in body temperature indicates an allergy to peanut.After isolating splenocytes from the peanut-allergic mice, use a one-milliliter insulin syringe equipped with a 27-gauge, 5/8-inch needle to intravenously inject 1.5 times 10 to the seventh of the extracted allergic splenocytes into the tail veins of naive, unsensitized animals. One day after adoptive transfer, intravenously inject 200 microliters of Ara h 2 antigenic liposomes into the tail vein of each BALB/c mouse that received the allergic splenocytes. Two weeks after the liposome injection, boost the mice with an i.p.injection of soluble Ara h 2, followed by a 200-microliter Ara h 2 challenge on day 61. Then, measure the body temperatures with the rectal probe every 15 minutes as demonstrated. Protein conjugation can be demonstrated by running a reducing gel showing an increase in molecular weight compared to the unconjugated protein.To assess the antigenic liposome-stimulated B-cell calcium flux, gate the live single cells to allow selection of the B220-positive CD5-negative B cells. The ratio of Indo-1 violet versus Indo-1 blue fluorescence over time can then be analyzed to assess the amount of liposome-induced B-cell activation. The quantification of Ara h 2-specific IgE and IgG1 in the serum of peanut extract-sensitized mice by ELISA indicates that mice with conferred sensitivity that are boosted with Ara h 2 exhibit measurable Ara h 2-specific IgE and IgG1 in their serum.Body temperatures recorded during the Ara h 2 challenge reveal that allergic mice demonstrate decreased body temperatures following the challenge, while body temperatures in naive mice remain consistent. While attempting this procedure, it's important to remember to carefully keep track of the amount of lipid-linked protein being incorporated into the liposomes, as too much protein can make extrusion difficult. Following this procedure, other methods like the tracking and sorting of allergen-specific B and T cells can be performed to answer additional questions about how these allergen-specific cells respond to therapies.This technique will pave the way for researchers in the allergy field to explore new immunotherapies that combat allergic disease using this mouse model. Don't forget that working with cholera toxin can be hazardous and that the guidelines for its use should be followed according to your local institutional biological safety standards.

Research

JoVE Journal

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Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

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Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

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Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

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Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

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Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

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Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...