我们要感谢罗斯实验室谁帮助优化效率最高和产量协议的前成员。

流感H3N2 VLP的产生1.真核表达系统<ol><li>亚克隆病毒结构的糖蛋白血凝素（HA），神经氨酸酶（NA），和人免疫缺陷病毒（HIV）的Gag的p55到真核表达载体中，如先前所描述pTR600 7 </li><li>扩增的DNA在化学感受大肠杆菌（ 例如 ，DH5α）和使用质粒纯化试剂盒按照制造商的说明分离转染级的质粒。的DNA的量依赖于产率和用户的需要。 </li><li>保持哺乳动物细胞系，293T，在含有10％胎牛血清（FBS）中于37℃，5％CO 2在加湿培养箱中生长培养基。 </li><li>生长的细胞，使得为T-150的组织培养烧瓶中含有每烧瓶45-75×10 6个细胞，使得烧瓶获取由第二天完整细胞粘附和85-95％的细胞融合。检查显微镜所需conflu下细胞ency。 注意:高细胞密度通常建议使用市售的转染试剂然而过度汇合可能导致副产物的细胞废物的积累和减少细胞生存力时，得到最小的细胞毒性最佳蛋白表达。 </li><li>以每个T-150烧瓶中40微克的总DNA量的Gag:在转染当天，制备脂质体和DNA的混合物作为根据制造商的建议，并转染DNA的细胞以1的DNA组合物:1:2的HA:NA。稀释在无血清转染培养基DNA和脂质体溶液不含抗生素，使得每个烧瓶中含有的20-30毫升的总体积。 注:基因结构的比例可能会要求用户进行优化。比例为1:虽然在这里没​​有证明，一个类似的转染过程一直与呼吸道合胞病毒（RSV）的F和HIV Gag的以1进行。对于DENV和CHIKV单一表达质粒，共有20微克每T-150瓶DNA的用于优化Ë的表达。 DNA:转染试剂的比例是用户进行了优化。使用推荐的转染培养基基于转染的方法， 即 ，聚阳离子脂质转染建议减少或缺失胎牛血清从而介质可以补充有生长激素和微量元素，以支持在无血清的生长。 </li><li>返回烧瓶至37℃，5％CO 2培养箱。为200毫升的体积，用9的T-150烧瓶中。将细胞保持在转染培养基直到VLP收获的那天。 </li><li>从视后72-96小时转染后的抗原，或当细胞活力下降至70-80％，通过显微镜检查所估计细胞收获培养上清液。转移上清液到50ml锥形管中并旋转该细胞向下，在500×g下5分钟，4℃以沉淀细胞碎片。 注意:3天上清液用新鲜的预热，无血清转染培养基到贴壁细胞将yiel的代用达第二批VLP的相似或略有减少产量，应由用户进行优化。 </li><li>池上清液过滤通过经由超速离心沉降之前0.22μm的微孔膜。 </li><li>测试用标准血凝测定14所述的HA表达的VLP用0.8％火鸡或哺乳动物红细胞的血细胞凝集活性或具有抗原特异性的ELISA进行。参见图2代表性的结果。 </li></ol> 2. CHIK VLP表达杆状病毒/昆虫细胞系统<ol><li>生成使用商业杆状病毒表达系统，从S-27菌株表达基孔肯雅病毒衣壳和包膜蛋白（C-E3-E2-6K-E1）重组杆状病毒。 </li><li>培养草地夜蛾 Sf9细胞在28℃用100单位/ ml青霉素和100μg/ ml链霉素的无血清的Sf9生长培养基中。用3 - 5×10 5℃/ ml的启动为suspens转瓶培养离子的细胞生长。通过经常当他们达到2-4×10 6℃/毫升（每3-4天）的细胞密度。 </li><li>文化Sf9细胞悬浮在旋转烧瓶在130转多点搅拌板系统上搅拌。为适当的通气性，维​​持培养物体积在旋转烧瓶的不超过一半的体积。 </li><li>对CHIK VLP的表达，感染Sf9细胞在以1的感染复数一的重组杆状病毒2×10 6个细胞/ ml的密度，并返回到28℃培养箱。典型地，感染一或两个转瓶，含有250毫升Sf9细胞。 注意:虽然我们不采用这种方法，昆虫细胞可以使用振荡器平台，在摇瓶培养是在28°C。 </li><li>收获培养物后72-96小时感染后，或者当细胞生存力降低至70-80％，用台盼蓝排阻根据制造商的协议确定的。 注:细胞的同时继续感染增殖和有一〜80％存活率在感染重组CHIK VLP杆状病毒3天感染后（dpi）的Sf9昆虫培养物。杆状病毒感染的细胞在外观上变大。形态也可能由圆形变为椭圆形。在杆状病毒感染的晚期阶段，细胞开始溶解。 </li><li>转移培养物直接从悬浮培养到50ml锥形管中并旋转该细胞向下在500 xg离心在4℃下5分钟。 </li><li>收集上清液并通过超速离心沉淀前通过0.22微米孔径的膜过滤器。 </li></ol> VLP / SVP的3沉淀/净化<ol><li>消毒对25mm×89毫米敞篷超速离心管并具有生物安全罩70％的乙醇。确保乙醇使用前已经擦干。 </li><li>最多可加载32毫升上清到一个干净的试管中。推荐25毫升最小音量，以防止塌陷和充满不足超速离心管的损坏。 </li><li>仔细下垫上清机智在PBSħ无菌3毫升20％甘油（V / V）。确保管是平衡的。 </li><li>在旋转XG 135000 4小时在4℃。 </li><li>吸上清，确保颗粒不从管打跑。 </li><li>重悬沉淀的VLP在用无菌PBS管的底部剧烈上下抽吸。 PBS的量所需的重悬的VLPs取决于VLP总蛋白产量和下游应用。通常情况下，每个悬浮颗粒VLP至少在100微升。 </li><li>商店样品4℃短期储存和-80℃下贮存用于长期存储。 </li></ol> 4.确定蛋白质产量和特异性抗原产量<ol><li>确定使用市售的蛋白定量方法，如BCA测定按照生产商的方案的总蛋白质含量。 </li><li>为了评估特定抗原含量，通过涂布标准抗原和2-5的系列稀释液进行直接的酶联免疫吸附试验（ELISA）1;上至ELISA 96孔平底板g总蛋白的样品。 <ol><li>与抗原特异性抗体按照探测在平板上抗原的存在，并使用常规的ELISA开发衬底以产生用于酶标仪检测吸光度。参见图2为样本HA-VLP表达HA和ELISA检测的代表性结果; HA和NA的多种组合是可能，但单产可能在HA-NA兼容15不同。 注意:抗原特异性抗体具有可变亲和力和建议的抗体的终点稀释滴定执行VLP量化之前确定。商业抗体将具有用于在ELISA中使用的建议的浓缩或稀释的范围，以及建议的协议。 </li></ol></li></ol>

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作者什么都没有透露。

我们使用以上描述的技术成功表达并纯化的SVP和各种病原体多种结构蛋白组成的VLP。在一般情况下，我们使用哺乳动物表达系统，以产生我们的VLP。然而，在我们的手中，哺乳动物细胞衍生CHIK VLP收益率较低。使用重组杆状病毒昆虫细胞系统时，CHIK VLP产量为更加强劲。在一般情况下，杆状病毒 - 昆虫细胞系统产生的同时控制下更高量的重组蛋白，这可能是由于，并且还由于高水平的外源基因的表...

病毒样颗粒（VLP）是人类疫苗接种核准技术。事实上，一些比较现代感许可的疫苗，包括人乳头瘤病毒（HPV）和肝炎Β的（HepΒ）疫苗采用这种方法。的VLP从能够自组装的结构蛋白形成。组装的粒子模仿病毒形态，但不能感染或复制，因为它们缺乏病毒基因组。的VLP可表达并从许多原核和真核系统的纯化。对文献的审查表明，不同的表达系统按以下标准使用:细菌- 28％，酵母- 20％，植物- 9％，昆虫- 28％，和哺乳动物- 15％1。值得注意的是，基于L1衣壳蛋白HPV疫苗是在酵母中（Gardasil的），或在昆虫细胞系统（的Cervarix）2制备。 HepΒ疫苗，Recombivax和的Engerix-Β，也产生了酵母和组成HepΒ表面抗原3的4。 我们使用哺乳动物和昆虫细胞表达系统来产生需要用于组装多个结构蛋白的共表达的VLP。我们的工作主要集中在设计，生产和净化基于VLP的疫苗对人类病原体:流感病毒，呼吸道合胞病毒（RSV），登革热病毒（DENV）和基孔肯雅病毒（CHIKV）。我们的方法是足够的灵活性，以允许从多个表达质粒的多种结构蛋白的共表达，或单个表达质粒（ 图1）。先前，我们制备并纯化的H5N1的VLP从编码人胚胎肾293T细胞5,6-流感血凝素（HA），神经氨酸酶（NA），和基质（M1）的质粒的共表达装配。用于在哺乳动物细胞中表达的基因进行密码子优化，并克隆到pTR600含有巨细胞病毒立即早期启动子加上内含子的用于启动创见一个真核表达载体真核刀片ription和转录7终止牛生长激素聚腺苷酸化信号。用于产生人类季节性流感亚型H3N2的VLP的本研究中使用的HA，NA（ 图1A）和另一种基质蛋白，HIV的Gag的p55，三个质粒共表达了类似的方法，并已被证明产生大小颗粒流感8种相似的病毒样颗粒。虽然流感疫苗主要诱导抗HA抗体，增加流感病毒神经氨酸酶介导唾液酸酶的活性，使VLP转染细胞的9，也提出了另外的免疫原性目标崭露头角。以产生的RSV的VLP，我们还选择的HIV Gag的的无关核心来设计呈现完全RSV表面糖蛋白，以进一步展示使用HIV Gag的VLP形成灵活性，如先前描述和表征通过电子显微镜6,10原型疫苗。其他先前已经表明，病毒样颗粒呈现呼吸道合胞病毒糖蛋白可以利用新城疫病毒（NDV）11和流感基质12多种病毒组件进行组装。全长表面糖蛋白的序列在本研究中利用保留天然构象可能是必要的功能性受体结合和抗体识别测定通过酶联免疫吸附试验（ELISA）。 我们使用单一质粒表达系统来生成颗粒的例子是DENV和CHIKV。在登革病毒的情况下，我们可以生产亚病毒颗粒（的SVP）从含有的prM / E的结构基因的表达盒13中的单个质粒中的293T细胞无衣壳。术语SVP用于表示在病毒结构蛋白的组装缺少核心或衣壳蛋白。 CHIK的VLP也可使用含有CHIKV结构基因盒，编码衣壳和包膜蛋白的单个质粒产生的，或在我NSECT细胞通过用重组杆状病毒编码相同的结构基因盒为昆虫细胞表达（ 图1B - C）的优化感染。 表达办法上面讨论的最终结果是VLP的释放到可随后通过超速离心通过20％甘油缓冲纯化细胞培养液中。在这份报告中，我们提出的方法来表达和哺乳动物和昆虫细胞系统净化这些病毒样颗粒。

<table><tbody><tr><td>Plasmid Maxi Kit</td><td>Qiagen</td><td>12163</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-013</td><td></td></tr><tr><td>Lipofectamine</td><td>Life Technologies</td><td>L3000075</td><td>Lipofectamine 2000, Lipofectamine 3000</td></tr><tr><td>Opti-MEM I</td><td>Life Technologies</td><td>31985062</td><td></td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Life Technologies</td><td>10359-016</td><td></td></tr><tr><td>Cimarec I 6 multipoint stirrer plate</td><td>ThermoFisher Scientific</td><td>50094596</td><td></td></tr><tr><td>Polyclear ultracentrifuge tubes</td><td>Seton</td><td>7025</td><td>Confirm appropriate tubes for ultracentrifuge and bucket size</td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23235</td><td></td></tr><tr><td>Phosphate citrate buffer</td><td>Sigma</td><td>P4922</td><td></td></tr><tr><td>o-phenylenediamine dihydrochloride&amp;nbsp;</td><td>Sigma</td><td>P8287</td><td></td></tr><tr><td>0.22 &amp;mu;m vacuum filter top (500 ml)</td><td>Nalgene</td><td>569-0020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>339741&amp;nbsp;</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>ThermoFisher Scientific</td><td>10902-096</td><td></td></tr></tbody></table>

expression and purification of virus-like particles for vaccination

病毒样颗粒（VLPs）和亚病毒颗粒（的SVP）是另一种方法，以病毒疫苗的设计，在使用活病原体提供了更高的生物安全性和稳定性的优点。非感染和自组装，的VLP被用来呈现结构蛋白作为免疫原，绕过活病原体或用于抗原递送的重组病毒载体的需要。在这篇文章中，我们证明VLP设计和开发的不同阶段的临床前动物试验未来的应用。该过程包括以下阶段:抗原的选择，在选择的细胞系的VLP / SVP的纯化抗原的表达，并量化为抗原剂量。我们演示如何使用哺乳动物和昆虫细胞系，为我们的抗原表达和展示方法产量的差异。提出的方法可以应用于各种病原体，并且可以通过替换与免疫原性str中的抗原来实现感兴趣的用户的微生物的uctural蛋白质。病毒样颗粒和的SVP协助抗原特性和最佳候选疫苗的选择。

VLP产量分别由病毒抗原结构的设计变量。在这个协议中，我们已经证明了在上清液生产SVP或VLP的纯化通过超速离心使用昆虫和哺乳动物细胞的。 DENV的prM / E的结构基因的表达盒的四种亚型被用来构建DENV的SVP（界定为1-4）的版本于表1，并在0.6毫升卷证明1.1-2.6毫克总蛋白的范围内。对于需要三个基因构建病毒样颗粒，我们发现1优化的DNA转染比:1:2的HA:NA:加格分?...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

这里，我们提出用于合成使用任一杆状病毒或哺乳动物表达系统，并超速离心纯化病毒样颗粒的协议。这种高度可定制的方式被用来识别在一个安全的，灵活的方式的病毒抗原作为疫苗的目标。

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

expression-and-purification-of-virus-like-particles-for-vaccination

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.8K 次观看.  University of Georgia. 这里，我们提出用于合成使用任一杆状病毒或哺乳动物表达系统，并超速离心纯化病毒样颗粒的协议。这种高度可定制的方式被用来识别在一个安全的，灵活的方式的病毒抗原作为疫苗的目标。 

视频: Expression and Purification of Virus-like Particles for Vaccination

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

科研

癌症研究

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

从尿囊液中生产高滴度重组新城疫病毒

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

免疫与感染

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

一种简单有效的方法构建突变牛痘病毒载体

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

生物工程

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

基于外膜囊泡显示SARS-CoV-2受体结合域的"即插即用"纳米颗粒疫苗平台

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Source: Feng, R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64213">A &#34;Plug-And-Display&#34; Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain.</a> J. Vis. Exp. (2022).
This video demonstrates a method to generate multivalent-displaying outer membrane vesicle (OMV) vaccines. Mammalian cells are transfected to express and secrete viral antigens fused to a SpyTag and a polyhistidine tag. These antigens are then purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. The purified antigen is mixed with outer membrane vesicles expressing surface-bound SpyCatcher (SC) protein. Multiple antigens covalently attach to SpyCatchers via their SpyTags (ST), creating OMVs that display the antigen, serving as a multivalent vaccine.

生成多价显示外膜囊泡疫苗

1. Plasmid construction

	Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (ClyA) plasmid (see Table ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Source: Arevalo, M. T., et al.&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/54041">Expression and Purification of Virus-like Particles for Vaccination.</a> J. Vis. Exp. (2016).
This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

一种通过哺乳动物系统生成流感病毒样颗粒的技术

Source: Arevalo, M. T., et al.&#160; Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).
This video demonstrates the ...