Name: a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies
Uploaded: 2018-02-23T12:30:00.000+00:00
Duration: 4 min 47 s
Description: Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

该项目的经费部分来自国家过敏和传染性疾病研究所、国家卫生研究所、卫生和人类服务部的联邦基金, CEIRS 合同 P01AI097092-04S1 (P.E.L.)。

本手稿中所做的实验是按照伊坎医学院的《道德和法规规范》进行的。
1. 通过转染或感染来表达流感病毒血凝素
染:
<ol><li>平板人胚胎肾脏 (HEK 293T) 细胞密度为 2 x 104细胞/在白色组织培养中处理96井板, 让它在4个°C 孵化器 (与 37 CO5%) 坐在 2 h。</li>
	<li>染细胞与 100 ng DNA 编码的病毒血凝素和 0.2 ul 的转染试剂每井在 50 ul 的总容量。 注: 质粒和 transfectant 试剂均在1x 最小必需培养基中稀释, 降低血清培养基。</li>
	<li>在37个°C 孵化器中孵化出 5% CO2为18小时的模板后, 删除转染介质。</li>
</ol>2. 感染
<ol><li>板 A549 或 Madin 达犬肾细胞密度为 2 x 104细胞/以及在白色组织培养处理96井板和增长至少 18 h 在37个°C 孵化器与 5% CO2。</li>
	<li>在37个°C 孵化器与 5% CO2中, 在1x 的内存中稀释病毒并感染细胞, 其感染的多样性为3、1或 0.33, 1 h。总容积应等于 100 ul。 注意: 10x 内存被水稀释, 使1x 内存工作库存用于上述病毒稀释. </li>
	<li>感染后, 取出接种, 并在没有胰蛋白酶的情况下加入 100 ul 1x 的记忆。</li>
	<li>在 5% CO2的 37 °C 孵化器中, 将受感染的细胞增长18到24小时。</li>
</ol>3. 通过单克隆抗体或血清评估 FcRg/NFAT 介导的荧光素酶活性的活化
<ol><li>首先, 用25µL 的检测介质 (1x RPMI 1640 补充 4% (卷/卷) 低 IgG 胎牛血清) 取代转染或感染细胞的生长培养基。</li>
	<li>在一个单独的96井板, 执行四倍稀释的抗体的兴趣开始在30微克/毫升使用化验媒介。在 triplicates 中执行稀释, 并在图 1A中演示一个示例板布局。请确保包含一个不包括抗体 (无抗体控制) 和背景控制 (单独检测缓冲区) 的控件。 注: 对于 "no 抗体" 和背景控制, 添加25µL 的检测缓冲器, 而不是抗体。</li>
	<li>将25级稀释抗体从步骤3.2 转移到转染细胞或受感染细胞的相应井上。</li>
	<li>孵育板在 37 °C 孵化器 (与 5% CO2) 为15分钟。</li>
	<li>添加 25 ul 的适当修改效应 Jurkat 细胞表达小鼠 FcgRIV 或人类 FcgRIIIa (7.5万细胞/井)。每个井的总容积应为75µL, 抗体的起始最终浓度为10µg/毫升 (图 1B)。 注意: 对于背景控制, 添加25µL 的检测缓冲区, 而不是效应细胞。为了检查抗体依赖性细胞介导的吞噬功能, 可以使用一种表达人 FcgRIIa 的修饰 Jurkat 细胞。</li>
	<li>孵化板在一个37°c 孵化器与 5% CO2为 6 h。</li>
	<li>解冻和温暖的荧光素酶基板缓冲在 37 °C 水约1小时前使用。 注: 为了使荧光素酶基板缓冲, 重建冻干荧光素酶测定基质在化验缓冲区提供。重构的基板缓冲区可存储在-30 °c 到-10 °c 上, 最多六周。</li>
	<li>添加 75 ul/井的荧光素酶基板缓冲除背景控制以外的所有水井。</li>
	<li>读一紫外线。</li>
	<li>用以下方程式计算褶皱感应: 折叠感应 = (诱发值-背景值)/(无单克隆值-背景值)。</li>
</ol>

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作者声明没有利益冲突。

此处描述的方法允许用户测量 HA 特定单克隆抗体与小鼠 FcγRIV 的接触能力。靶抗原、流感病毒血凝素, 在病毒感染或质粒 DNA 转染后在细胞表面表达。这项试验还可以与其他 FcγRs (人类或小鼠) 结合使用不同的表面表达的病毒蛋白。此外, 我们使用血清样本来评估抗体的能力, 在多克隆的情况下, 以诱发 Fc 效应函数 (数据未显示), 这可能提供更多的相关信息, 当研究感染诱发的适应性免疫反应或接种.我们还推测, 该检测可以被修改, 以测量抗体的诱导识别内部病毒蛋白直接涂膜的可溶性蛋白。然而, 这种方法还没有经过严格的测试。
抗体依赖效应函数的诱导可能受多种因素的影响, 包括但不限于效应器目标比 (每井增加的效应细胞数), 使用的抗体浓度, 潜伏期之间的孵化时间效应因子和靶细胞, 以及在检测缓冲区中使用的低 IgG 血清浓度。
从历史上看, 检测抗体依赖性细胞细胞毒性活性的标准检测方法包括来自捐赠者的主要自然杀伤细胞。FcγR 的参与和细胞内通路的活化通常由激活标记 CD107a 和/或干扰素γ19,23,24,31的表达式决定。虽然经典的化验方法使用更多相关的效应细胞, 因为他们还没有基因工程, 捐助者的变化, 由于在 FcγR 的等位基因差异可能会影响 NK 细胞活化, 并导致批次变异。替代性化验作为 ADCC 活动的代理人, 包括 FcγR 二聚体的 ELISA 或铬释放杀死试验25。尽管使用 FcγR 二聚体的 ELISA 是高通量的, 但由于此种方法不使用效应细胞32, 这种检测可能与生理学无关。虽然铬释放试验措施直接杀死, 它不是高吞吐量, 并要求原 NK 细胞再次是捐助者依赖33。最近的一种方法描述了使用小鼠 T 细胞杂交瘤稳定表达嵌合体 FcγR-CD3ζ链分子和措施活化通过分泌的 IL-2。所描述的分析类似于此处描述的方法, 它利用修改后的 T 细胞作为表示奇异激活 FcγR34的效应细胞的代理。另外, 表达 FcγRIIIa 的细胞线 (NK92.05 CD16) 可以作为效应细胞, 脱粒抗原 CD107a 的上调作为活化标记使用。然而, 病毒和病毒肽在酶联免疫吸附膜上的应用应注意, 因为涂在疏水性表面的蛋白质可能并不总是重述抗原35的生理结构。
这里描述的方法是96井格式化验, 利用生理上相关的和本机表达的病毒靶点, 可以修改为小鼠或人的抗体。基因改良的 Jurkat 细胞可以保持在文化和保存的36。虽然目前的效应细胞只表达一个激活 FcγR, 在测量抗体依赖细胞介导的效应函数时, 在本研究中所描述的使用本协议的许多优点都应考虑。

季节性流感疫苗提供的免疫传统上是通过血凝抑制 (HI) 检测方法1来评估的, 它测量了针对血凝素受体结合部位的抗体的存在。这些高活性抗体可以赋予杀菌免疫, 但在其保护范围内一般是狭窄的, 只对少数几株流感病毒提供免疫力。广泛反应性单克隆抗体的分离和鉴定血凝素 (HA) 表明, 普遍流感疫苗的发展范围内可达2, 3, 4,5,6,7,8. 普遍流感疫苗的主要目标之一是对流感病毒的保守区域(9、10、11、12 )产生强烈的抗体应答。,13,14,15,16. 有广泛的反应性抗体识别这些保守的表位, 由中和和非中和抗体17,18, 被证明需要 Fc FcγR 的相互作用, 以优化保护在体内并突出显示 Fc 介导的免疫对流感病毒的整体免疫应答的贡献19,20。
保护的相关性对于评估感染的免疫力至关重要。这些指标允许科学家和临床医生评估疫苗的功效、数据的重要性和治疗过程。预防流感病毒感染的唯一确定的相关性是血凝抑制试验。1:40 的滴度与患病风险的50% 降低有关 (121 ), 并反映了抗体的存在, 通过靶向位于 HA 球状头的受体结合点来抑制凝集。然而, 也应该指出, T 细胞反应可能是更好的相关性预防流感病毒感染的老年人。有趣的是, 最近的一份报告表明, 神经氨酸酶抑制活性可能是更好的预测流感免疫的22。幸运的是, 典型的体外检测方法, 如中和或神经氨酸酶抑制试验可以测量 HA 茎或神经氨酸酶特异抗体。然而, 大多数这些常规的体外化验只考虑抗体的抗原结合区的功能, 而不测量 Fc 区域的作用。此外, 未检测到通过 Fc 受体参与在体内中保护的非中和抗体的贡献17,18。为了测量诱发 Fc 介导免疫的抗体所提供的保护, 如抗体依赖性细胞介导的毒性 (ADCC), 需要一个健壮的体外检测。
下面描述的方法评估小鼠单克隆抗体的能力, 通过使用基因修饰的 Jurkat 细胞系, 表达激活鼠型 1 FcR, FcγRIV, 诱导 Fc 介导的功能。抗体参与 FcγR transduces 细胞内信号, 触发核因子的活化 T 细胞介导的荧光素酶活性。该方法优于传统技术, 需要分离和培养原效应细胞, 使用流式细胞仪检测 Fc 介导的效应函数的激活19,23,24 ,25,26。首先, 这里描述的协议可以很容易地被改编成包含不同病毒的目标, FcγRs 和不同物种 (人和小鼠) 的抗体。其次, 使用修饰的 Jurkat 细胞系表达 FcγR 与荧光素酶报告基因在活化 T 细胞 (NFAT) 核因子下, 允许一个大的格式分析, 可以很容易地用一个平板阅读器测量发光。在这里, 传统的激活的主要效应细胞被取代诱导 NFAT-荧光素酶的抗体接触 FcγR 表面上的 Jurkat 细胞。这96井板格式化验允许测量多达四个不同的样品在 triplicates 与七稀释-许多样品可能是繁琐的使用传统技术。另外还有一点需要考虑, 这可能会影响实验设计和数据的解释。病毒靶必须在细胞表面表达, 以便抗体识别。为了减轻这种情况, 目标抗原 (例如内部病毒蛋白) 可以直接涂在盘子的表面上。然而, 这还没有经过严格的测试。此外, 修改后的 Jurkat 细胞系只表达一种激活 FcγR 的类型, 不表达任何抑制 FcγRs, 而主细胞系表达所有受体在生理环境中。
我们以前用这种方法来证明多克隆语境中的表位特异性在调节 Fc 介导的效应函数方面起着关键作用, 而抗体依赖性细胞介导反应的最佳活化需要两点联系27,28。在这里, 我们描述一个方法, 评估的能力, 茎特定单克隆抗体参与和激活小鼠激活 FcγRIV。尽管有异常29、30, 但大量广泛的反应性抗体瞄准了血凝素的抗原保守的茎区, 从而在普遍流感的发展中起着重要作用。疫苗.

<table><tbody><tr><td>A549 cells&amp;nbsp;</td><td>ATCC</td><td>CCL-185</td><td>Adenocarcinomic human alveolar basal epithelial cells</td></tr><tr><td>HEK 293T cells</td><td>ATCC</td><td>CRL-3216</td><td>Human embryonic kidney cells</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies&amp;nbsp;</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>1X Opti-MEM Reduced Serum Medium</td><td>ThermoFisher Scientific</td><td>51985-034</td><td></td></tr><tr><td>White tissue culture treated 96-well plate</td><td>Corning, Inc.&amp;nbsp;</td><td>3917</td><td>Assay plates</td></tr><tr><td>10X MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>Jurkat cell expressing murine FcgRIV</td><td>Promega</td><td>M1201</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIIa</td><td>Promega</td><td>G7010</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Jurkat cell expressing human FcgRIIa</td><td>Promega</td><td>G9901</td><td>Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum</td></tr><tr><td>Luminometer</td><td>BioTek</td><td>Synergy H1 Multi-Mode reader</td><td>Luminescence plate reader</td></tr><tr><td>Bio-Glo Luciferase Assay System</td><td>Promega</td><td>G7940</td><td>Contains luciferase assay buffer and luciferase assay substrate</td></tr><tr><td>Madin Darby canine kidney cells</td><td>ATCC</td><td>CCL-34</td><td>Canine kidney cells</td></tr></tbody></table>

a method to assess fc-mediated effector functions induced by influenza hemagglutinin specific antibodies

抗体通过其抗原结合域和 Fc 区域, 在耦合病毒病原体的先天和适应性免疫应答中起着至关重要的作用。在这里, 我们描述如何测量 Fc 效应函数的活化作用的单克隆抗体靶向流感病毒血凝素与使用基因工程的 Jurkat 细胞系表达活化型 1 Fc FcγR. 使用此方法,免疫球蛋白所赋予的特定 Fc-FcγR 相互作用的贡献可以通过体外测定来确定。

我们证明, 一个茎特定的单克隆, 6F12, 但不是头部特异的单克隆, PY102, 能够激活主要的自然杀伤细胞植入 CD107a 和干扰素γ19。为了模拟茎特异性抗体激活原发性 NK 细胞的能力, 我们表明, 一个茎特异性的单克隆抗体, 6F12, 可以有力地激活修饰的 Jurkat 细胞, 表达小鼠 FcγRIV 14 倍。另一方面, 头部特定的单克隆抗体, PY102 和对照 IgG 不 (图 2)28。
为了调查感染的多样性 (Jurkat) 是否会影响表达小鼠 FcγRIV 的细胞的诱导, 我们感染 MDCK 细胞 X31 (重组流感病毒表达血凝素和神经氨酸酶/红 Kong/1/68 (H3N2)病毒与 A/波多黎各 Rico/8/34 (甲型h1n1 流感) 的内部段), 其教学语言为3、1或0.33。二十四小时后, Jurkat 细胞表达小鼠 FcγRIV 和单克隆9H10 被添加到受感染的 MDCK 细胞。在图 3中, 我们显示感染了3语言的细胞可以诱导3倍以上的发光, 而 MDCK 细胞感染了0.33 的语言。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56256/56256fig1.jpg"> 图 1: 样本板布局和实验组组成示意图.(A) 每个抗体样本的起始浓度为10µg/毫升, 并在板上连续稀释 (1 列到12列) 4 倍。每个样品都是在 triplicates 中完成的。为 no 单克隆控件保留行 G, 行 H 为背景。(B) 每个实验组的最终体积, 然后再加入荧光素酶基板缓冲物75µL。值得注意的是, 样本和 ' no 单克隆组 ' 有25µL 的效应细胞增加, 而背景对照组有75µL 的化验媒介。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56256/56256fig2.jpg"> 图 2: 通过茎单克隆抗体诱导修饰的 Jurkat 细胞表达 FcgR.A549 细胞感染了 A/波多黎各 Rico/8/34 (甲型h1n1 流感), 其教学语言为3。24小时后感染, 添加了小鼠 FcγRIV 表达 Jurkat 细胞与 PY102, 6F12 或 IgG 控制。用萤火虫荧光素酶在 NFAT 反应元素下产生的发光信号对 Jurkat 细胞的诱导进行评价。误差条 = 标准偏差。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56256/56256fig3.jpg"> 图 3: 感染的多样性会影响茎特异抗体的诱导.MDCK 细胞感染了 X31 (H3N2), 其教学语言为3、1或0.33。在 24 h 后感染, FcgR 表达 Jurkat 细胞结合 9H10 (开始稀释10µg/毫升) 添加。用萤火虫荧光素酶在 NFAT 反应元素下产生的发光信号对 Jurkat 细胞的诱导进行评价。误差条 = 标准偏差。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56256/56256fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies at JoVE.com

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

我们描述了一种方法来衡量 Fc 介导的效应函数的活化作用的抗体, 针对流感病毒血凝素。这种检测也可以适应评估单克隆抗体或多克隆血清靶向其他病毒表面糖蛋白, 以诱导 Fc 介导的免疫能力。

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

a-method-to-assess-fc-mediated-effector-functions-induced-influenza

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.6K 次观看.  Icahn School of Medicine at Mount Sinai. 我们描述了一种方法来衡量 Fc 介导的效应函数的活化作用的抗体, 针对流感病毒血凝素。这种检测也可以适应评估单克隆抗体或多克隆血清靶向其他病毒表面糖蛋白, 以诱导 Fc 介导的免疫能力。

视频: A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

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

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

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 ...

科研

免疫与感染

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

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

医学

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

测量 HA 抗体对 Fc 介导的效应子功能的激活

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Source: Leon, P. E., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56067">Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for generating escape variants of neutralizing influenza virus monoclonal antibodies. Antibody-neutralized virus suspension is injected into embryonated chicken eggs to propagate escape variants selectively. This results in a virus population predominantly of escape variants, confirmed via a Hemagglutination assay.

针对中和抗体的流感病毒逃逸变异的产生

NOTE: HA-specific antibodies that inhibit viral replication can generally be categorized into i) ones that bind on or adjacent of the receptor binding ...

The Hemagglutination Inhibition Assay to Detect Serum Antibodies Against a Target Antigen

Source: Kaufmann, L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55833">An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.</a> J. Vis. Exp. (2017)
The video demonstrates a hemagglutination inhibition assay for detecting serum antibodies against specific antigens. Confirmation of hemagglutination inhibition in the test well and hemagglutination in the control well is achieved through distinct visual patterns. This assay plays a crucial role in assessing immune response, vaccine effectiveness, and studying viral infections in virology and immunology.

用于检测针对靶抗原的血清抗体的血凝抑制试验

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...