抗原脂质体用于生成特定于疾病的抗体
1Janssen R&D, 2Department of Microbiology and Immunology,University of North Carolina, 3UNC Food Allergy Initiative,University of North Carolina, 4Department of Pediatrics,University of North Carolina, 5Department of Chemistry,University of Alberta, 6Department of Molecular Medicine,Scripps Research Institute, 7Department of Medical Microbiology and Immunology,University of Alberta
Summary
描述了抗原脂质体纳米粒子的制备及其在体外和体内刺激 B 细胞活化的作用。一致和稳健的抗体反应导致了一个新的花生过敏模型的发展。抗原脂质体的生成协议可以扩展到不同的抗原和免疫模型。
Abstract
抗体反应为各种病原体提供了重要的保护性免疫力。仍然有很高的兴趣, 产生强大的疫苗接种抗体, 以及了解致病抗体反应如何发展在过敏和自身免疫疾病。生成强健的抗原特异抗体反应并不总是微不足道的。在小鼠模型中, 它通常需要用佐剂进行多轮免疫接种, 从而导致诱导抗体水平的变化很大。一个例子是在小鼠花生过敏模型, 其中更健壮和可重现的模型, 最小化鼠标数和使用佐剂将是有益的。这里介绍的是一个高度可重现的小鼠花生过敏性过敏症模型。这个新模型依赖两个关键因素: (1) 抗原特异性脾细胞过继转输从花生敏化小鼠转移到天真的受体小鼠, 使大量小鼠抗原特异记忆 B 和 T 细胞的数量正常化;(2) 随后以脂质体纳米颗粒显示主要花生过敏原 (Ara h 2) 的形式, 对受体小鼠进行了强多价抗原的增强。这个模型的主要优点是它的重现性, 最终降低了每项研究中使用的动物数量, 同时最大限度地减少了接受多重注射佐剂的动物数量。这些免疫原性脂质体的模块化组装提供了相对较容易适应其他过敏或自身免疫的模型, 涉及致病抗体。
Introduction
食物过敏影响了美国8% 的儿童, 并且在过去十年中增加了1的患病率。对花生过敏影响1% 儿童, 通常不超过2。虽然一些有前途的临床试验正在进行的治疗食物过敏, 包括口服免疫疗法 (OIT), 舌下免疫治疗 (狭缝) 和 epicutaneous 免疫治疗 (EPIT), 目前没有 FDA 批准的治疗策略对于脱敏花生过敏的个人3,4,5,6,7,8。因此, 过敏个体必须严格避免过敏原, 以避免过敏反应。许多问题仍然是关于敏感的途径和食物过敏发展的根本机制。
小鼠模型是研究过敏机制以及开发新的耐受性和脱敏疗法9,10,11,12的宝贵工具。这是特别正确的, 因为主要的花生过敏原 (艾拉 h 2;Ah2) 在人类也是主要的过敏原在几个描述的鼠标模型13,14。虽然小鼠花生过敏模型在研究敏感度和耐受性机制方面是非常宝贵的, 但缺点是它们可以是可变的, 需要使用佐剂。更有效的免疫原将是减少此类模型固有变异性的一种方法。由于 b 细胞是由多价抗原强烈激活, 显示过敏原的抗原脂质体是一个很好的选择, 因为他们有能力通过 b 细胞受体 (BCR) 激活 b 细胞, 同时也具有有效的属性通过非特异的抗原呈现细胞来启动 T 细胞室。
在这里, 我们描述了一个详细的协议共轭蛋白抗原脂质体纳米粒子使用一个简易的和模块化的策略。通过使用替代抗原, 抗 IgM Fab 片段, 我们证明了这种抗原脂质体在刺激 B 细胞活化中的作用。采用抗原脂质体显示 Ah2 抗原, 研制出一种新的小鼠赋敏模型。在这个模型中, 脾细胞从证实的花生过敏小鼠, 含有花生特异记忆 B 和 T 细胞, 被转移到天真的同类系小鼠。记忆抗体反应是通过注射脂质体与 Ah2 结合在受体小鼠中诱发的, 以诱导抗体对 Ah2。其次只有一个促进与可溶性 Ah2, Ah2-specific 抗体引起强烈的过敏反应, 当这些小鼠随后与 Ah2 挑战。由于过敏反应的小鼠反应以高度一致的方式, 并没有得到佐剂, 这种方法是一个可取的花生过敏模型, 结果表明, 它可能有效用在其他鼠标模型驱动的抗原导向过敏原和可能自身抗原。
Protocol
将蛋白质与脂质结合在一起的一般方法主要基于早期的工作15。下面描述的所有动物程序都已通过北卡罗来纳大学教堂山机构动物护理和使用委员会 (IACUC) 批准。在花生过敏模型中使用的所有小鼠都是 BALB/cJ 女性从3周的年龄购买。阿尔伯塔大学动物护理和使用委员会 (ACUC) 已经批准了实验, 涉及使用小鼠脾进行体外分析, 从 C57Bl/6 小鼠至少6周的年龄。 1. …
Representative Results
与 DSPE (2000) 的兴趣蛋白的共轭可以通过运行减少显示与游离蛋白相比, 分子量的增加来证明。图 1A显示了抗鼠 IgM F (ab) 片段共轭 DSPE 的代表性凝胶, 它显示了变性蛋白的 kDa bandshift。请注意, 大约50% 的蛋白质似乎被修改, 这是预期的, 因为1:1 化学计量学是在单体碎片, 这是一个沉重的和轻链的一个。图 1B显示了 Ah2 共轭到 PEG-DSPE ?…
Discussion
这里概述的方法是一个通用的协议的蛋白质共轭的脂质, 使在脂质体纳米颗粒的蛋白质显示。对于非常大的多亚基蛋白质, 此协议可能有有限的效用。理想的方法是引入一个特定于站点的标签, 使之能够使用双正交化学连接策略。如果表达蛋白质制备, 这可以使用可用的特定于站点的策略17, 和广泛的功能组在聚乙二醇脂质的末端是商业可用的。因此, 本议定书的目的是将与天然来?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国防部 (W81XWH-16-1-0302 和 W81XWH-16-1-0303) 的赠款的支持。
Materials
| Model 2110 Fraction Collector | BioRad | 7318122 | |
| Cholestrol | Sigma | C8667 | Sigma grade 99% |
| SPDP | Thermo Fisher Scientific | 21857 | |
| DSPC | Avanti | 850365 | |
| DSPE-PEG 18:0 | Avanti | 880120 | |
| DSPE-PEG Maleimide | Avanti | 880126 | |
| Extruder | Avanti | 610000 | 1mL syringe with holder/heating block |
| Filters 0.1 µm | Avanti | 610005 | |
| Filters 0.8 µm | Avanti | 610009 | |
| 10mm Filter Supports | Avanti | 6100014 | |
| Glass Round Bottom Flask | Sigma | Z100633 | |
| Turnover stoppers | Thermo Fisher Scientific | P-301398 | |
| Tubing | Thermo Fisher Scientific | P-198194 | |
| Leur Lock | Thermo Fisher Scientific | k4201634503 | |
| Sephadex G50 Beads | GE Life Sciences | 17004201 | |
| Sephadex G100 Beads | GE Life Sciences | 17006001 | |
| Heat Inactivated Fetal Calf Serum | Thermo Fisher Scientific | 10082147 | |
| HEPES (1M) | Thermo Fisher Scientific | 15630080 | |
| EGTA | Sigma | E3889 | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
| 1x RBC lysis Buffer | Thermo Fisher Scientific | 00-4333-57 | |
| Indo-1 | Invitrogen | I1203 | |
| CD5-PE | BioLegend | 100608 | |
| B220-PE-Cy7 | BioLegend | 103222 | |
| HBSS | Thermo Fisher Scientific | 14170112 | without calcium and magnesium |
| MgCl2 | Sigma | M8266 | |
| CaCl2 | Sigma | C4901 | |
| Fab anti-mouse IgM | Jackson ImmunoResearch | 115-007-020 | |
| F(ab')2 anti-mouse IgM | Jackson ImmunoResearch | 115-006-020 | |
| Peanut flour | Golden Peanut Co. | 521271 | 12% fat light roast, 50% protein |
| Animal feeding needles | Cadence Science | 7920 | 22g x 1.5", 1.25 mm – straight |
| Microprobe thermometer | Physitemp | BAT-12 | |
| Rectal probe for mice | Physitemp | Ret-3 | |
| Cholera toxin, from vibrio cholera | List Biological Laboratories, Inc. | 100B | Azide free |
| BCA Protein Assay Kit | Pierce | 23225 | |
| Carbonate-bicarbonate buffer | Sigma | C3041 | |
| TMB Stop Solution | KPL | 50-85-06 | |
| SureBlue TMB Microwell Peroxidase Substrate | KPL | 5120-0077 | |
| 96 well Immulon 4HBX plate | Thermo Scientific | 3855 | |
| Purified soluble Ara h 2 | N/A | N/A | purified as in: Sen, et al., 2002, Journal of Immunology |
| HSA-DNP | Sigma | A-6661 | |
| Mouse IgE anti-DNP | Accurate Chemical | BYA60251 | |
| Sheep anti-Mouse IgE | The Binding Site | PC284 | |
| Biotinylated Donkey anti-Sheep IgG | Accurate Chemical | JNS065003 | |
| NeutrAvidin Protein, HRP | ThermoFisher Scientific | 31001 | |
| Mouse IgG1 anti-DNP | Accurate Chemical | MADNP105 | |
| HRP Goat anti-mouse IgG1 | Southern Biotech | 1070-05 | |
| 1 mL Insulin Syringes | BD | 329412 | U-100 Insulin, 0.40 mm(27G) x 16.0 mm (5/8") |
| Superfrost Microscope Slides | Fisher Scientific | 12-550-14 | 25 x 75 x 1.0 mm |
| ACK Lysing Buffer | gibco by Life Technologies | A10492-01 | 100 mL |
| RPMI 1640 Medium | Thermo Fisher Scientific | 11875093 | 500 mL |
| Cell Strainer | Corning | 352350 | 70 μm Nylon, White, Sterile, Individually packaged |
| NuPAGE 4-12% Bis-Tris Protein Gels | Invitrogen | NP0322BOX | 10 gels |
| NuPAGE LDS buffer, 4X | Invitrogen | NP0008 | 250 mL |
| SeeBlue Plus2 Pre-stained standard | Invitrogen | LC5925 | 500 µL |
| NuPAGE MES/SDS running buffer, 20X | Invitrogen | NP0002 | 500 mL |
| GelCode Blue Stain | Thermo Scientific | 24590 | 500 mL |
References
- Gupta, R. S., et al. The prevalence, severity, and distribution of childhood food allergy in the United States. Pediatrics. 128 (1), e9-e17 (2011).
- Sicherer, S. H., Munoz-Furlong, A., Godbold, J. H., Sampson, H. A. US prevalence of self-reported peanut, tree nut, and sesame allergy: 11-year follow-up. Journal of Allergy and Clinical Immunology. 125 (6), 1322-1326 (2010).
- Kim, E. H., et al. Sublingual immunotherapy for peanut allergy: clinical and immunologic evidence of desensitization. Journal of Allergy Clinical Immunology. 127 (3), 640-646 (2011).
- Vickery, B. P., et al. Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy. Journal of Allergy and Clinical Immunology. 133 (2), 468 (2014).
- Jones, S. M., et al. Epicutaneous immunotherapy for the treatment of peanut allergy in children and young adults. Journal of Allergy and Clinical Immunology. 139 (4), 1242 (2017).
- Varshney, P., et al. A randomized controlled study of peanut oral immunotherapy: clinical desensitization and modulation of the allergic response. Journal of Allergy Clinical Immunology. 127 (3), 654-660 (2011).
- Anagnostou, K., et al. Assessing the efficacy of oral immunotherapy for the desensitisation of peanut allergy in children (STOP II): a phase 2 randomised controlled trial. Lancet. 383 (9925), 1297-1304 (2014).
- Sampson, H. A., et al. Effect of Varying Doses of Epicutaneous Immunotherapy vs Placebo on Reaction to Peanut Protein Exposure Among Patients With Peanut Sensitivity: A Randomized Clinical Trial. The Journal of the American Medical Association. 318 (18), 1798-1809 (2017).
- Bednar, K. J., et al. Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model. Journal Immunology. 199 (9), 3116-3128 (2017).
- Macauley, M. S., et al. Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis. Journal Clinical Investigation. 123 (7), 3074-3083 (2013).
- Orgel, K. A., et al. Exploiting CD22 on antigen-specific B cells to prevent allergy to the major peanut allergen Ara h 2. Journal Allergy Clinical Immunology. 139 (1), 366-369 (2017).
- Smarr, C. B., Hsu, C. L., Byrne, A. J., Miller, S. D., Bryce, P. J. Antigen-fixed leukocytes tolerize Th2 responses in mouse models of allergy. Journal of Immunology. 187 (10), 5090-5098 (2011).
- Kulis, M., et al. 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. Clinical and Experimental Allergy. 42 (2), 326-336 (2012).
- Dang, T. D., et al. Increasing the accuracy of peanut allergy diagnosis by using Ara h 2. Journal of Allergy Clinical Immunology. 129 (4), 1056-1063 (2012).
- Loughrey, H. C., Choi, L. S., Cullis, P. R., Bally, M. B. Optimized procedures for the coupling of proteins to liposomes. Journal Immunological Methods. 132 (1), 25-35 (1990).
- Sen, M., et al. Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes. Journal of Immunology. 169 (2), 882-887 (2002).
- Krall, N., da Cruz, F. P., Boutureira, O., Bernardes, G. J. Site-selective protein-modification chemistry for basic biology and drug development. Nature Chemistry. 8 (2), 103-113 (2016).
- Jimenez-Saiz, R., et al. Lifelong memory responses perpetuate humoral TH2 immunity and anaphylaxis in food allergy. Journal Allergy and Clinical Immunology. 140 (6), 1604-1615 (2017).
- Moutsoglou, D. M., Dreskin, S. C. Prolonged Treatment of Peanut-Allergic Mice with Bortezomib Significantly Reduces Serum Anti-Peanut IgE but Does Not Affect Allergic Symptoms. International Archives of Allergy and Immunology. 170 (4), 257-261 (2016).
- LaMothe, R. A., et al. Tolerogenic Nanoparticles Induce Antigen-Specific Regulatory T Cells and Provide Therapeutic Efficacy and Transferrable Tolerance against Experimental Autoimmune Encephalomyelitis. Frontiers in Immunology. 9, 281 (2018).
- Srivastava, K. D., et al. Investigation of peanut oral immunotherapy with CpG/peanut nanoparticles in a murine model of peanut allergy. J Allergy Clin Immunol. 138 (2), 536-543 (2016).
- Bellinghausen, I., Saloga, J. Analysis of allergic immune responses in humanized mice. Cellular Immunology. 308, 7-12 (2016).
- Pillai, S., Mattoo, H., Cariappa, A. B cells and autoimmunity. Curr Opin Immunol. 23 (6), 721-731 (2011).
- Mantegazza, R., Cordiglieri, C., Consonni, A., Baggi, F. Animal models of myasthenia gravis: utility and limitations. International Journal of General Medicine. 9, 53-64 (2016).
- Berman, P. W., Patrick, J. Experimental myasthenia gravis. A murine system. J Exp Med. 151 (1), 204-223 (1980).
- Berman, P. W., Patrick, J. Linkage between the frequency of muscular weakness and loci that regulate immune responsiveness in murine experimental myasthenia gravis. J Exp Med. 152 (3), 507-520 (1980).
- Derksen, V., Huizinga, T. W. J., van der Woude, D. The role of autoantibodies in the pathophysiology of rheumatoid arthritis. Seminars in Immunopathology. 39 (4), 437-446 (2017).
Cite This Article
Bednar, K. J., Hardy, L., Smeekens, J., Raghuwanshi, D., Duan, S., Kulis, M. D., Macauley, M. S. Antigenic Liposomes for Generation of Disease-specific Antibodies. J. Vis. Exp. (140), e58285, doi:10.3791/58285 (2018).