Opsono-坚持性检测，以评估疫苗开发中针对 无烟杆菌 和其他封装病原体的功能抗体
Summary
opsono-依从性测定是分析抗体在疫苗开发中的opsonic功能的替代方法。
Abstract
蛋白石粘性检测是一种功能性检测，它列举了细菌病原体与专业噬细胞的附着。因为坚持是法戈西病和杀戮的必要条件，因此测定是奥普索诺-法细胞杀菌检测的替代方法。opsono-依从性检测的一个优点是可以选择使用灭活病原体和哺乳动物细胞系，从而允许跨多个实验进行标准化。在检测中使用灭活病原体也有助于生物安全3级传染性剂和其他致命病原体的工作。在我们的工作中，opsono-依从性检测用于评估抗体的功能能力，从用炭热胶囊疫苗免疫的动物血清，到诱导固定 杆菌无烟杆菌 与小鼠大噬细胞系RAW 264.7的粘合。自动荧光显微镜用于捕捉巴西利粘合巨噬细胞的图像。增加依从性与血清中是否存在抗胶囊抗体有关。表现出高血清抗胶囊抗体浓度的非人类灵长类动物受到保护，免受炭热挑战。因此，蛋白石粘性检测可用于阐明血清中抗原特异性抗体的生物功能，评估疫苗候选物和其他治疗方法的疗效，并作为免疫力的可能相关性。
Introduction
病原体的识别、坚持、内化和退化是噬菌体细胞增多症1的组成部分，噬菌体是伊利亚·梅奇尼科夫在1883年第2、3年首次描述的宿主先天免疫反应的显著途径。噬细胞白细胞以及免疫系统的其他细胞在选择靶点时具有高度歧视性：他们能够区分”传染性非自我”和”非传染性自我”通过病原体相关的分子模式，通过他们的模式识别受体（PRR）4，5的剧目。宿主识别病原体也可能发生与宿主蛋白的结合，如补充和抗体6。这个过程，称为opsoning，用这些分子涂覆病原体，在与噬菌体细胞6的opsonic受体（例如补充和Fc受体）结合后增强内化。病原体要粘附在噬细胞上，就必须将多种受体与其同质配体集体结合。只有这样，坚持才能触发和维持主机单元内的信号级联，以启动内化6。
由于噬细胞病在清除病原体和预防感染方面的重要性，细胞外病原体已经开发出许多方法来颠覆这一过程，以延长其生存时间。一种重要的策略是生产一种离子聚合物（例如多糖或多氨基酸）胶囊，这种胶囊由于其电荷而具有抗噬菌性，免疫力差，并且保护细菌包络上的分子免受PRR6、7的侵害。病原体，如隐球菌新福尔曼和肺炎链球菌有由糖化物组成的胶囊，而葡萄球菌表皮和一些杆菌物种产生多ɣ谷氨酸（PGGA）7，8。然而，其他病原体产生的胶囊类似于非传染性自我。例如，链球菌和B.cereus的致病菌株有一个透明质酸胶囊，它不仅是抗噬菌体，而且可能不被免疫系统9，10识别为异物。
胶囊与载体蛋白的结合，将他们从贫乏的T独立抗原转化为高致免疫性T依赖抗原，可诱导高血清抗胶囊抗体滴度11，12。该策略用于针对肺炎、流感嗜血杆菌和奈塞里亚脑膜肽11的许可疫苗。抗胶囊抗体的手术活动通常由奥普索诺-法果细胞杀伤性检测（OPKA）13、14、15、16进行评估。这些检测测试功能抗体是否可以触发噬细胞增多症和杀死14。然而，使用带有传染性病原体的OPKA，如一级生物选择剂和毒素（BSAT），包括B.无烟体17，是危险的，并带来安全风险：这些检测需要广泛处理选择的代理。选择剂处理只能在受限生物安全级别 3 （BSL-3） 实验室进行：由于必须遵循许多安全和安保预防措施，这些领域的工作需要长期的操作程序。BSL-3实验室通常也没有配备用于OPKA工作的专用设备，如显微镜和细胞计。因此，我们开发了一种基于使用灭活细菌18，19的替代检测方法。我们称之为一种不依赖于内化和杀戮的opsono-依从性检测（OAA），作为检测输出：相反，对非活化灭活病原体的坚持被用作噬细胞病的指数。机械上，OAA是一个合适的替代品，因为坚持发生先验，并与内化和细胞内杀密切交织在一起。从生物安全的角度来看，OAA 是首选，因为它需要最少的传染性剂处理，实验时间比 OPKA 短，并且可以在 BSL-2 实验室中进行，在产生和转移灭活病原体库存后进行。
我们证明利用OAA来检查在非人类灵长类动物（NHPs）的血清中发现的抗胶囊抗体的opsonic功能，这种抗胶囊抗体是用胶囊结合而接种的[即从B.无烟菌结合到奈西里亚脑膜肽的外膜蛋白复合物（OMPC）的PGGA]20。血清通配的巴西利被孵育与附体鼠标巨噬细胞线，RAW 264.7。固定后，细胞单层和附体巴西利通过荧光显微镜成像。与对照血清20相比，当用用胶囊结合接种的NHPs的血清孵化时，细菌依从性增加。坚持与炭热挑战20，21的生存有关。因此，OAA的使用具有抗胶囊抗体的功能，大大方便了疫苗候选剂的测试。
Protocol
根据《动物福利法》、《公共卫生服务政策》和其他有关动物和涉及动物的实验的联邦法规和条例，此处描述的研究是根据机构动物护理和使用委员会批准的议定书进行的。进行这项研究的设施由国际实验室动物护理评估和认证协会认可，并遵守2011年国家研究理事会《实验室动物护理和使用指南》中规定的原则。 1. 细胞系的培养和维护，RAW 264.7 <p class="jov…
Representative Results
本节显示了在OAA实验中收集的具有代表性的显微图，以及显示OAA可用于检查抗体的生物功能的结果。在这里，该检测被成功地用于评估炭热疫苗候选的疗效。验证囊体上的封装状态至关重要，因为很少或根本没有封装会导致它们粘附在宿主细胞上，从而产生高背景。 图 1 是 B. 无烟艾 姆斯的图像，上面印有印度墨水，以检查封装。OAA 要求照亮多个相邻的视场，如果使…
Discussion
胶囊疫苗已被证明对许多细菌病原体有效，许多疫苗获准用于人类25、26、27。这些疫苗通过产生针对胶囊的抗体而起作用，其中许多研究使用OPKA来显示抗体13、14、16、28、29的蛋白细胞功能。出于对生物?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
蔡美利、查博特和弗里德兰德设计了手稿中描述的程序。蔡美利和T·普特蒙-泰勒进行了实验。D. 查博特进行了数据分析。蔡先生写了手稿。
作者感谢凯尔·菲茨出色的技术援助。
这项工作得到了国防威胁减少局授予煤层气的支持。VAXBT.03.10.RD.015，计划编号921175。
意见、解释、结论和建议是作者的意见、解释、结论和建议，不一定得到美国陆军的赞同。本出版物的内容不一定反映国防部的观点或政策，也不提及贸易名称、商业产品或组织，暗示美国政府的认可。
Materials
| 0.20 µm syringe filter (25mm, regenerated cellulose) | Corning, Corning, NY | 431222 | |
| 10 mL syringe (Luer-Lok tip) | BD, Franklin Lakes, NJ | 302995 | |
| 15µ 96 well black plates (plate #1 for imaging) | In Vitro Scientific, Sunnyvale, CA | P96-1-N | |
| 16% paraformaldehyde | Electron Microscopy Science, Hatfield, PA | 15710 | |
| 75 cm sq. tissue culture treated flask | Corning, Corning, NY | 430641 | |
| Agar (powder) | Sigma-Aldrich, St. Louis, MO | A1296 | |
| Baby Rabbit Complement | Cedarlane Labs, Burlington, NC | CL3441 | |
| Bacto Yeast Extract | BD, Sparks, MD | 288620 | |
| BBL Brain Heart Infusion (BHI) | BD, Sparks, MD | 211059 | |
| Blood Agar (TSA with Sheep Blood) plates | Remel, Lenexa, KS | R01198 | |
| Cell scraper | Sarstedt, Newton, NC | 83.183 | |
| Costar 96 well cell culture plates (plates #2 & 3 for dilutions) | Corning, Corning, NY | 3596 | |
| Cover glass | Electron Microscopy Science, Hatfield, PA | 72200-10 | |
| Difco Nutrient Broth | BD, Sparks, MD | 234000 | |
| Dulbecco's Modified Eagle Medium (DMEM), high glucose | Gibco, Thermo Fisher Scientific, Waltham, MA | 11965-092 | contains 4500 mg/L glucose, 4 mM L-glutamine, Phenol Red |
| EVOS FL Auto Cell Imaging System (fluorescence microscope) | Life Technologies, Thermo Fisher Scientific, Waltham, MA | AMAFD1000 | |
| Fetal Bovine Serum | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SH30071.03 | not gamma irradiated, not heat inactivated |
| Fluorescein isothiocyanate | Invitrogen, Thermo Fisher Scientific, Waltham, MA | F143 | |
| HCS Cell Mask Orange Cell Stain | Invitrogen, Thermo Fisher Scientific, Waltham, MA | H32713 | |
| hemocytometer (Improved Neubauer) | Hausser Scientific, Horsham, PA | 3900 | |
| India Ink solution | BD, Sparks, MD | 261194 | |
| L- glutamine (200 mM) | Gibco, Thermo Fisher Scientific, Waltham MA | 25030081 | supplement medium with additional 2mM L-glutamine |
| Nikon Eclipse TE2000-U (inverted compound microscope) | Nikon Instruments, Melville, NY | TE2000 | |
| PBS without Calcium or Magnesium | Lonza, Walkersville, MD | 17-516F | |
| Penicillin-Streptomycin Solution, 100x | Hyclone, GE Healthcare Life Sciences, South Logan, UT | SV30010 | |
| petri dishes (100 x 15 mm) | Falcon, Corning, Durham, NC | 351029 | for agar plates |
| RAW 264.7 macrophage cell line (Tib47) | ATCC, Manassas, VA | ATCC TIB-71 | |
| Slides | VWR, Radnor, PA | 16004-422 | |
| Sodium Bicarbonate | Sigma-Aldrich, St. Louis, MO | S5761 | |
| Trypan Blue Solution (0.4%) | Sigma-Aldrich, St. Louis, MO | T8154 | |
| Zeiss 700 Laser Scanning Microscopy (confocal microscope) | Carl Zeiss Microimaging, Thornwood, NY | 4109001865956000 |
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Citar este artículo
Chua, J., Chabot, D. J., Putmon-Taylor, T., Friedlander, A. M. Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens. J. Vis. Exp. (159), e60873, doi:10.3791/60873 (2020).