重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫
Summary
There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.
Abstract
Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies.
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.
Introduction
免疫记忆在没有疾病的产生是有效的疫苗接种1的生理基础。最近，系统生物学为基础的方法已经表明，成功的疫苗，如黄热病疫苗，诱导强的诱导先天免疫反应和活化的树突细胞（DC），几种亚群这反过来，导致多向活化的抗原特异性T细胞2,3。由于区议会是唯一的免疫细胞群，激活抗原特异性幼稚T细胞4的能力，其功能的疫苗接种过程中的研究是至关重要的理解免疫反应的疫苗，并设计未来战略打击有挑战性的病原体。
一种系统，允许在免疫应答的疫苗不同的DC子集的跟踪是需要的，以便建立直流偏移的精确动力学到淋巴组织，并因此提供洞察负责疫苗的特异性适应性免疫启动的生理机制。反向遗传学为基础的方法提供以产生修改的可能性，减毒活疫苗，可通过实验用于此目的。因为它的实现对流感的研究，基于质粒的反向遗传学已被广泛采用，以产生重组流感病毒株包括LAIVs。标准协议来拯救重组流感病毒所需要的多转染的含有八个流感病毒节段，以及扩增在一个宽松的系统，如的Madin-Darby犬肾高度转染的细胞系的双义质粒（产生正的和负义RNA）（ MDCK）细胞和/或鸡胚5。然而，反向遗传学中的应用，以产生为了研究疫苗的免疫机制的分子工具仍然未开发。
发电新的小鼠模型允许免疫细胞亚群，包括DCS具体枯竭，开辟了新的可能性，以了解潜在的疫苗引发保护的基本免疫机制。在小鼠和人类DC亚群功能之间的比较表明，在很大的程度上，小鼠和人DCs是功能性同源6,7-，这些发现，有力地表明，小鼠模型的发展允许DC的在稳定状态下的特定耗尽并且在炎性病症，可能有助于理解直流反应在人体中的生理。近年来已经产生了许多小鼠模型携带转基因的表达猿猴白喉毒素（DT）的受体（DTR）的兴趣8,9的基因的启动子区的控制之下。由于小鼠组织中不自然地表达DTR，这些模型允许携带鼠标时接种DT感兴趣的靶基因细胞亚群的条件枯竭。因此，我们的abiliTY过程中的生理过程耗尽特定DC和白细胞等在体内 ，已经大大的DTR-RO为主的发展增强。然而，虽然这些转基因小鼠模型已被广泛使用，以了解免疫系统的个体发育，其应用到疫苗开发已几乎没有测试。这里，通过结合流感反向遗传学和DTR基于竞争的骨髓嵌合体中，我们提出在疫苗的免疫应答的体内研究疫苗免疫以及个别基因功能的动力学的方法。这种技术的对具有挑战性的传染病新型疫苗临床前评价中的应用可以帮助理顺疫苗设计和体内测试候选疫苗。
Protocol
Representative Results
Discussion
在这项研究中，我们描述了如何反向遗传学和嵌合小鼠模型可用于阐明疫苗诱导的免疫的生理和分子机制。流感反向遗传学是建立在许多实验室，并发挥在了解发病的流感，复制和传输17主要作用。在我们的协议的一个关键点是表达外源抗原决定冷适应流感疫苗抢救。同时引入短的cDNA进神经氨酸苷酶的茎的策略已经由许多组，研究者需要确保没有另外的突变过程中的疫苗生产蛋和该疫苗?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Sergio Gómez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.
Materials
| Dulbecco´s Modified Eagle Medium (DMEM 1X) | Gibco RL-Life Technologies | 41965-039 | |
| Opti MEM | Gibco RL-Life Technologies | 31985-047 | |
| Lipofectamine 2000 | Invitrogen-Life Technologies | 11668-027 | |
| Penicillin-Streptomycin (10.000 U/ml) | PAA | p11-010 | |
| Bovine Serum Albumin | Sigma-Aldrich | A2153 | |
| Embryonated eggs | Valo biomedia Gmbh | ||
| PBS (1X) | Sigma-Aldrich | D8537 | |
| 70 μM Nylon Filters | Greiner-Biorad | 542-070 | |
| Red Blood Cell Lysing buffer (RBCL) 10X | BD Bioscience | 555899 | |
| CD16/CD32 Mouse BD Fc Block (2.4G2) | BD Pharmigen | 553142 | |
| APC-Anti-mouse SIINFEKL-H2kb (25 D1.16) | Biolegend | 141605 | |
| PE-Anti-mouse CD11c (HLA3) | BD Biosciences | 553802 | |
| eFluor 450-Anti-mouse MHCII (Md/114.15.2) | eBioscience | 48-5321-82 | |
| Pe-Cy7-Anti-mouse CD11b (M1/70) | Biolegend | 101216 | |
| PerCp/Cy5.5-Anti-mouse CD103 (2E7) | Biolegend | 121416 | |
| PE-Anti-mouse CD45.1 (A20) | eBioscience | 12-0453-82 | |
| V500-Anti-mouse CD45.2 (1O4) | BD Bioscience | 562130 | |
| PerCp-eFluor710 -Anti-mouse CD8a (53-6.7) | eBioscience | 46-0081-80 | |
| APC-Cy7-Anti-mouse CD3ε (145-2611) | Biolegend | 100325 | |
| eFluor450-Anti-mouse CD4 (GK 1.5) | eBioscience | 48-0041-80 | |
| CFSE Proliferation dye | eBioscience | 65-0850-85 | |
| Baytril 2.5% | Bayer | 65-0850-85 | |
| Dymethil-Sulfoxide (DMSO) | Sigma-Aldrich | D2650 | |
| Ovalbumin | Molecular probes | O23020 | |
| Diphteria Toxin (DT) | Sigma-Aldrich | D0564 | |
| Trypsin-TPCK | Sigma-Aldrich | T1426 | |
| BD FACsCanto II Flow cytometer | BD Biosciences | ||
| FlowJo cell analysis software 9.5 | Flowjo inc. | ||
| Trypan Blue Stain (0.4%) | Life technologies | T10282 | |
| Countess Automatic Cell Counter | Invitrogen-Life Technologies | C10227 |
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