宿主病原学反应及疫苗效果评价
Summary
在这里, 我们提出了一个优雅的协议, 在体内评估疫苗的有效性和宿主免疫反应。该协议可适用于研究病毒、细菌或寄生虫病原体的疫苗模型。
Abstract
疫苗是20世纪医学奇迹 。它们大大降低了传染病造成的发病率和死亡率, 并促使全球预期寿命显著延长。尽管如此, 确定疫苗的疗效仍然是一个挑战。新出现的证据表明, 目前的无细胞疫苗 (apv) 为百日咳杆菌 (百日咳 b.) 诱导次优免疫。因此, 一个主要的挑战是设计下一代疫苗, 在不产生全细胞疫苗 (wpv) 不利副作用的情况下诱导保护性免疫。在这里, 我们描述了一个协议, 我们用来测试一个有前途的, 新颖的佐剂的疗效, 扭曲免疫反应的保护性 thenth17 表型, 并促进更好地清除 b .百日咳挑战从小鼠呼吸道。本文介绍了小鼠免疫、细菌接种、组织采集和免疫反应分析的方案。利用这种方法, 在我们的模型中, 我们成功地阐明了一种有希望的下一代无细胞百日咳疫苗所引发的关键机制。该方法可应用于任何传染病模型, 以确定疫苗疗效。
Introduction
疫苗是20世纪最伟大的公共卫生成就之一, 但我们仍然没有完全理解成功疫苗刺激保护性免疫的机制。疫苗接种后诱导的分子特征 (如细胞激活标记、细胞亚型的扩展和基因表达模式) 的鉴定为预测和生成有效的信息提供了大量信息。免疫反应。在体外细胞培养系统1中无法充分复制宿主病原体反应的复杂性。体内疫苗模型旨在同时评估宿主内的多种免疫细胞类型。这提供了一个优势, 当描述疫苗抗原处理和呈现, 差异细胞因子分泌, 和扩大免疫细胞的特点。这里描述的协议提供了一个详细的方法来确定疫苗的疗效, 通过评估系统和局部免疫反应和量化病原体负担的有关组织。这里提供的例子测试了百日咳博德氏杆菌 (百日咳 b. b.)实验疫苗的有效性。
百日咳是一种革兰氏阴性细菌, 是呼吸道疾病百日咳 (百日咳)2,3的病因。与受感染的人密切接触 (有症状或无症状) 会导致传播、定植和疾病。尽管全球疫苗覆盖率很高, 但百日咳在世界许多国家被认为是一种重新出现的疾病, 是可预防的儿童死亡的主要原因 5,6,7,8. 2015年,百日咳和百日咳被列入国家过敏和传染病研究所 (niaid) 新出现的传染性病原病/疾病清单, 强调需要开发一种更好的疫苗, 使其长寿保护免疫。
目前, 控制百日咳复发的一个活跃的研究领域是开发下一代无细胞百日咳疫苗 (apv), 该疫苗具有新型佐剂和抗原的最佳组合, 以模拟全细胞产生的免疫反应百日咳疫苗 (wpv)9。使用所描述的协议, 我们最近报告说, 通过添加一种新型佐剂 bordetella 殖民因子 a (bcfa), 对目前 fda 批准的 apv 进行了修改, 从而更有效地减少了百日咳细菌的负荷。鼠肺10,11。这种增加的保护伴随着对更具保护性的 ththth这家免疫剖面10的铝诱导的 ththuth2 免疫反应的倾斜。该议定书是详细和全面的, 使调查员能够通过同时评估宿主和免疫对各种病原体的反应, 获得最大限度的信息。
这里描述的协议遵循具有代表性的疫苗计划, 如图 1所示, 以确保最佳的宿主免疫反应。
Protocol
所有活体动物实验都是根据俄亥俄州立大学 iacuc 根据 iacuc 的指导方针批准的协议进行的。c57bl6 小鼠被用于所有免疫接种和感染。根据国家卫生研究院的指导, 每组都使用雄性和雌性小鼠。根据实验组结果的预测差异, 通过功率计算确定了每组动物的数量。例如, 每组8只小鼠在α= 0.05 (双面) 时产生80% 的功率,进行2样本 t-测试, 以检测1.33 标准差 (sds) 的兴趣结果的差异。 1. ?…
Representative Results
所描述的模型显示了一种评估疫苗效率和宿主-病原体相互作用过程中免疫反应的方法。图 1描述了用于免疫和感染小鼠和采集组织进行分析的代表性疫苗计划。图 2显示了用于诱导小鼠的麻醉系统的设置, 使研究人员能够进行免疫接种和细菌接种。图 3显示了 od600的示例测量和计算, 以实现 1 od 细菌…
Discussion
这里描述的研究疫苗诱导的百日咳感染免疫的综合方案也将允许评估宿主对各种其他病原体的反应。该协议讨论了提供免疫接种的方法, 确定疫苗在病原体挑战后的疗效, 以及免疫功能的平行解剖。为了研究其他病原体, 在调整该议定书时, 需要修改几个参数。这些措施包括但不限于动物麻醉的方式、疫苗组成、剂量和给药途径。此外, 受质疑的病原体的剂量和给药途径、收获所选组织和免疫…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了俄亥俄州立大学10112556-01 兰特和启动资金的支持。
Materials
| 2L induction chamber | Vet Equip | 941444 | |
| Fluriso | Vet One | V1 501017 | any brand is appropriate |
| Bordet Gengou Agar Base | BD bioscience | 248200 | |
| Casein | Sigma | C-7078 | |
| Casamino acids | VWR | J851-500G | Strainer Scholte (SS) media components |
| L-Glutamic acid | Research Products Int | G36020-500 | |
| L-Proline | Research Products Int | P50200-500 | |
| Sodium Chloride | Fisher | BP358-10 | |
| Potassium Phosphate monobasic | Fisher | BP362-1 | |
| Potassium Chloride | Fisher | P217-500 | |
| Magnesium Chloride hexahydrate | Fisher | M2670-500G | |
| Calcium Chloride | Fisher | C75-500 | |
| Tris base | Fisher | BP153-1 | |
| L-cysteine HCl | Fisher | BP376-100 | SS media suplements |
| Ferrous Sulfate heptahydrate | Sigma | F-7002 | |
| Niacin | Research Products Int | N20080-100 | |
| Glutathione | Research Products Int | G22010-25 | |
| Ascorbic acid | Research Products Int | A50040-500 | |
| RPMI 1640 | ThermoFisher Scientific | 11875093 | |
| FBS | Sigma | F2442-500mL | any US source, non-heat inactivated |
| gentamicin | ThermoFisher Scientific | 15710064 | |
| B-mercaptoethanol | Fisher | BP176-100 | |
| 15mL dounce tissue grinder | Wheaton | 357544 | any similar brand is appropriate |
| Cordless Hand Homogenizer | Kontes/Sigma | Z359971-1EA | any similar brand is appropriate |
| Instruments – scissors, curve scissors, forceps, fine forceps, triangle spreaders | any brand is appropriate | ||
| 3mL syringes | BD bioscience | 309657 | |
| 15mL conical tubes | Fisher | 339651 | |
| 1.5mL microfuge tubes | Denville | C2170 | |
| 70um cell strainers | Fisher | 22363548 | |
| 60mm plates | ThermoFisher Scientific | 130181 | |
| 48-well tissue culture plates | ThermoFisher Scientific | 08-772-1C | |
| 1mL insulin syringe 28G1/2 | Fisher Scientific/Excel Int. | 14-841-31 | |
| Mouse IFN-gamma ELISA Ready-SET-Go! Kit | Invitrogen / eBioscience | 50-173-21 | |
| Mouse IL-17 ELISA Ready-SET-Go! Kit | Invitrogen / eBioscience | 50-173-77 | |
| Mouse IL-5 ELISA Ready-SET-Go! Kit | Invitrogen / eBioscience | 50-172-09 |
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Caution, K., Yount, K., Deora, R., Dubey, P. Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice. J. Vis. Exp. (144), e58930, doi:10.3791/58930 (2019).