评估纳米乳剂佐剂疫苗对耐甲氧西林 金黄色葡萄球菌 （MRSA）感染的免疫反应
Summary
本协议制备和评估新型纳米乳剂佐剂疫苗的物理特性、免疫应答和 体内 保护作用。
Abstract
纳米乳佐剂疫苗因其粒径小、热稳定性高、有效诱导免疫应答的能力等特点而受到广泛关注。然而，建立一系列全面的方案来评估新型纳米乳剂佐剂疫苗的免疫反应至关重要。因此，本文采用严格的程序来确定疫苗的理化特性（通过透射电子显微镜 [TEM]、原子力显微镜 [AFM] 和动态光散射 [DLS]）、疫苗抗原和系统的稳定性（通过高速离心机测试、热力学稳定性测试、SDS-PAGE 和蛋白质印迹）和特异性免疫应答（IgG1， IgG2a 和 IgG2b）。使用这种方法，研究人员可以 准确评估新型纳米乳剂佐剂疫苗在致死MRSA252小鼠模型中的保护作用。通过这些方案，可以确定在有效佐剂潜力方面最有希望的纳米乳疫苗佐剂。此外，这些方法可以帮助优化新型疫苗的未来开发。
Introduction
耐甲氧西林 金黄色葡萄球菌 （MRSA） 是一种机会性病原体，是全球重症监护病房 （ICU）1 病房、心脏病科和烧伤科感染率最高的病原体之一。MRSA表现出较高的感染率，死亡率和广泛的耐药性，在临床治疗中存在很大的困难2。在世界卫生组织（WHO）于2017年发布的抗生素耐药细菌全球优先清单中，MRSA被列为最关键的第3类。因此，迫切需要针对MRSA感染的疫苗。
铝助剂使用时间较长，佐剂辅助机理比较明确，安全有效，耐受性好4.铝助剂是目前广泛使用的佐剂类型。一般认为吸附在铝盐颗粒上的抗原可以提高注射部位的稳定性，增强吸收抗原的能力，提供良好的吸收和缓释5。目前，铝佐剂的主要缺点是它们缺乏佐剂作用或仅对某些候选疫苗抗原表现出弱佐剂作用6。此外，铝佐剂诱导IgE介导的超敏反应5。因此，有必要开发新型佐剂来刺激更强的免疫反应。
纳米乳液助剂是由油、水、表面活性剂和助表面活性剂7组成的胶体分散体系。此外，佐剂具有热力学稳定性和各向同性，可通过高速离心高压灭菌或稳定，并且可以在温和的制备条件下自发形成。目前市场上有几种乳液助剂（如MF59、NB001-002系列、AS01-04系列等），但其粒径大于160nm8。因此，纳米级（1-100 nm）药物制剂的优势（即大比表面积、小粒径、表面效应、高表面能、小尺寸效应和宏观量子隧穿效应）无法得到充分利用。在本协议中，据报道，一种基于纳米乳液技术的新型佐剂具有直径尺寸为1-100nm的佐剂，其具有良好的佐剂活性9。我们测试了重组亚基疫苗抗原蛋白HI（α-溶血素突变体[Hla]和Fe离子表面决定因子B[IsdB]亚基N2活性片段融合蛋白）;建立了一系列程序来检查疫苗的物理性质和稳定性，评估其肌内给药后的特异性抗体反应，并使用小鼠全身感染模型测试疫苗的保护作用。
Protocol
动物实验根据实验动物使用和护理手册进行，并得到第三军医大学实验动物福利与伦理委员会的批准。6-8周龄的雌性Balb / c小鼠用于本研究。这些动物是从商业来源获得的（见 材料表）。 1. MRSA HI抗原蛋白的制备 从商业来源获得IsdB和Hla克隆（见材料表），进行聚合酶链反应（PCR）扩增以扩增IsdB和Hla基因，并将…
Representative Results
评估了制备纳米乳液佐剂疫苗的方案以及该疫苗的体外和体内测试。TEM、AFM 和 DLS 用于确定该样品表面 zeta 电位和粒径的重要特征（图 1）。SDS-PAGE和蛋白质印迹显示，离心后沉淀物和上清液中的抗原量未显著降解，表明疫苗结构完整、特异性和免疫原性（图2）。纳米乳佐剂疫苗显著提高了总IgG、IgG1和IgG 2a抗体滴度。疫苗的免疫原性显着?…
Discussion
IsdB是一种细菌细胞壁锚定和铁调节的表面蛋白，在获得血红素铁的过程中起重要作用15。 Hla，α毒素，是MRSA中已知的最有效的细菌毒素之一，可以在真核细胞中形成孔隙并干扰粘附和上皮细胞16。本研究基于 IsdB 和 Hla抗原基因，利用基因工程技术构建并表达新型重组MRSA抗原蛋白（HI）。然后，采用低能量乳化法研制出纳米乳化疫苗，…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由国家重点研发计划2021YFC2302603、国家自然科学基金项目项目32070924号32000651号、重庆市自然科学基金项目项目2019jcyjA-msxmx0159支持。
Materials
| 5424-Small high speed centrifugeFA-45-24-11 | Eppendorf, Germany | 5424000495 | |
| 96-well plates | Corning Incorporated, USA | CLS3922 | |
| AFM Dimension FastScan | BRUKER, Germany | null | |
| Alcohol lamp | Shenzhen Yibaxun Technology Co.,China | YBS-AA-11408 | |
| Balb/c mice | Beijing HFK Bioscience Co. Ltd. | ||
| BCIP/NBT | Fuzhou Maixin Biotechnology Development Company,China | BCIP/NBT | |
| Bio-Rad 6.0 microplate reader | Bio-Rad Laboratories Incorporated Limited Co., CA, USA | null | |
| BL21 Competent Cell | Merck millipore,Germany | 70232-3CN | |
| BSA-100G | Sigma-Aldrich, USA | B2064-100G | |
| Centrifuge 5810 R | Eppendorf, Germany | 5811000398 | |
| Coomassie bright blue G-250 staining solution | MIKX,China | DB236 | |
| Decolorization solution | BOSTER,China | AR0163-2 | |
| Electro-heating standing-temperature cultivator HH-B11-420 | Shanghai Yuejin Medical Device Factory, China | null | |
| Electrophoresis apparatus | Beijing Liuyi Instrument Factory, China | DYCZ-25D | |
| Gel image | Tanon, USA | null | |
| Glutathione-Sepharose Resin GST | Mei5bio,China | affinity chromatography resin | |
| H2SO4 | Chengdu KESHI Chemical Co., LTD,China | 7664-93-9 | |
| HI recombinant protein | Third Military Medical University,China | 110-27-0 | |
| HRP -Goat Anti-Mouse IgG | Biodragon, China | BF03001 | |
| HRP- Goat anti-mouse IgG1 | Biodragon, China | BF03002R | |
| HRP- Goat anti-mouse IgG2a | Biodragon, China | BF03003R | |
| HRP- Goat anti-mouse IgG2b | Biodragon, China | BF03004R | |
| Inoculation loop | Haimen Feiyue Co.,LTD,China | YR-JZH-1UL | |
| IsdB and Hla clones | Shanghai Jereh Biotechnology Co,China | null | |
| Isopropyl nutmeg (pharmaceutic adjuvant) | SEPPIC, France | null | |
| isopropyl- β-D-1-mercaptogalactopyranoside | fdbio,China | FD3278-1 | |
| LB bouillon culture-medium | Beijing AOBOX Biotechnology Co., LTD,China | 02-136 | |
| Lnfrared physiotherapy lamp | Guangzhou Runman Medical Equipment Co.,China | 7600 | |
| Low temperature refrigerated centrifuge | Eppendorf, Germany | null | |
| Malvern NANO ZS | Malvern Instruments Ltd., UK | null | |
| MH(A) medium | Beijing AOBOX Biotechnology Co., LTD,China | 02-051 | |
| MH(B) medium | Beijing AOBOX Biotechnology Co., LTD,China | 02-052 | |
| Micro plate washing machine 405 LSRS | Bio Tek Instruments,Inc Highland Park,USA | null | |
| Mini-TBC Compact Film Transfer Instrument | BeiJingDongFangRuiLi Co.,LTD,China | 1658030 | |
| MMC packing | TOSOH(SHANGHAI)CO.,LTD | 0022818 | |
| MRSA252 | USA, ATCC | null | |
| Nanodrop ultraviolet spectrophotometer | Thermo Scientific, USA | null | |
| New FlashTM Protein any KD PAGE Protein electrophoresis gel kit | DAKEWE, China | 8012011 | |
| PBS | biosharp, China | null | |
| PCR, Amplifier | Thermal Cycler, USA | null | |
| pGEX-target gene recombinant plasmid | Shanghai Jereh Biotechnology Co,China | B3528G | |
| Phosphotungstic acid | G-CLONE, China | CS1231-25g | |
| pipette | Eppendorf, Germany | 3120000844 | |
| polyoxyethylated castor oil (pharmaceutic adjuvant) | Aladdin, China | K400327-1kg | |
| Primary antibody | Laboratory homemade:from immunized mice with positive sera | null | See Reference 11 for details |
| propylene glycol (pharmaceutic adjuvant) | Sigma-Aldrich, USA | P4347-500ML | |
| Protein Marker | Thermo Scientufuc, USA | 26616 | |
| PVDF TRANSFER MEMBRANE | Invitrogen,USA | 88518 | |
| Scanning Electron Microscope | JEOL,Japan | JSM-IT800 | |
| Sodium pentobarbital | Merck,Germany | Tc-P8411 | |
| Talos L120C TEM | Thermo Fisher, USA | null | |
| TMB color solution | TIAN GEN, China | PA107-01 | |
| Turtle kits | Xiamen Bioendo Technology Co.,LTD | ES80545 | |
| Tween-20 | Macklin, China | 9005-64-5 |
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Cite This Article
Zeng, X., Sun, H., Ye, Y., Luo, X., Cai, D., Yang, Y., Chen, T., Sun, C., Zhang, S., Zeng, H. Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection. J. Vis. Exp. (199), e65152, doi:10.3791/65152 (2023).