Name: intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity
Uploaded: 2015-06-25T13:00:00
Description: Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

We thank Sergio G&#243;mez-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.

动物实验按照批准的方案和下列德国动物保护法的准则进行的。所有开展动物实验的工作人员根据B类或欧洲实验动物科学协会联合会了C通过培训计划。 1.重组产生流感减毒活疫苗通过反向遗传学注：对于重组流感病毒通过反向遗传学产生的具体协议已经由先前的研究5和超出本报告的范围。简单地说，冷适应流感疫苗（CAV）的救助是在生物安全II?...

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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?...

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。

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			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
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			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
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			<td>Biolegend</td>
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			<td>BD Bioscience</td>
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			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
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			<td>eBioscience</td>
			<td>65-0850-85</td>
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			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
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			<td>Dymethil-Sulfoxide (DMSO)</td>
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			<td>Ovalbumin&#160;</td>
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			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
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			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
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			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
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			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
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			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
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			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
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intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

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.

重组减毒活流感疫苗的产生可通过质粒双向启动子5的控制下编码流感病毒的八个段的转染来实现。冷适应流感疫苗通常含有冷适应株以及HA和NA选择的流感病毒株（ 如 H1N1）（ 图1A）的六个部分。冷适应的原则在33℃，在小鼠和人类14上层呼吸道（URT）的温度是基于病毒限制复制。因此，疫苗可以复制到在​​URT某种程度上但不能引起下呼吸道肺炎。在病毒神经?...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

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.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

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 ...

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

协议RBD - SARS疫苗的重组蛋白的制备，动物疫苗接种和中检测

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

鼻咽癌相关淋巴组织（NALT）在鼠标的响应中的作用研究疫苗

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...