这项工作得到了乔治梅森大学内部研究基金的支持。

1. 病毒和病毒颗粒组装
<ol><li>载体：以商业方式购买 SARS-CoV-2 M、E 或 N 以及 SARS-CoV-2 S（野生型，Wt）、Delta （B.1.617.2） 和 Omicron （B.1.1.529） 的表达载体。 注意：表达载体的蛋白质序列在 补充文件1中提供。这些表达向量的供应商也可以在 材料表下找到。</li>
	<li>细胞和细胞培养：在含有 10% 热灭活胎牛血清 （FBS）、50 单位/mL 青霉素和 50 μg/mL 链霉素的 Dulbecco 改良 Eagle 培养基 （DMEM） 中维持 HEK293T 细胞。 注意：所有细胞培养工作都需要在层流生物安全柜中完成。聚乙烯亚胺 （PEI） 转染通常需要 5-6 小时孵育。需要事先仔细考虑开始时间。</li>
	<li>病毒组装和基于 PEI 的共转染：通过HEK293T细胞共转染组装 Ha-CoV-2 颗粒。在共转染前一天，在 10 mL 完全 DMEM 培养基中将细胞接种在 10 cm 细胞培养皿（每个培养皿 4-5 x 106 个细胞）中。在这项研究中，使用三个培养皿分别用于接种用于组装 Ha-CoV-2 野生型、Delta 和 Omicron 的细胞。</li>
	<li>将培养皿在37°C的CO2 培养箱中孵育过夜。 第二天早上检查培养皿，确保细胞80%汇合。取出完全培养基，并用 9 mL DMEM 无血清培养基替换。</li>
	<li>对于每个培养皿，制备共转染混合物，其中包含 2.5 μg SARS-CoV-2 结构蛋白表达载体（N、E、M）、10 μg pAlphaPro-Luc-GFP-PreΨ（HaCoV2 基因组）和 2.5 μg S 蛋白表达载体（Delta 或 Omicron S 变体）和 45 μL 基于 PEI 的转染试剂。通过孵育 13 分钟（孵育时间不要超过 30 分钟）让共转染混合物形成复合物。</li>
	<li>孵育后，将共转染混合物缓慢地逐滴加入每个培养皿中。将培养皿置于37°C的CO2 培养箱中6小时。6小时后，除去无血清DMEM，并用完全DMEM培养基代替。共转染后48-60小时收获病毒。</li>
	<li>病毒收集和储存：共转染后 48 小时收获颗粒。通过在单层表面反复移液分离细胞，并从每个培养皿中收集细胞，放入15mL离心管中，并以400× g 离心5分钟。收集上清液并通过0.22μM过滤器。将Ha-CoV-2假病毒储存在-80°C。</li>
</ol>2.病毒感染性测定
<ol><li>细胞和细胞培养：在含有 10% 热灭活 FBS、50 单位/mL 青霉素和 50 μg/mL 链霉素的 Dulbecco 改良 Eagle 培养基 （DMEM） 中维持 HEK293T （ACE2/TMPRSS2） 细胞。</li>
	<li>接种 HEK293T （ACE2/TMPRSS2） 细胞：在病毒感染性测定的前一天，将 HEK293T（ACE2/TMPRSS2） 细胞接种在 96 孔板中的 50 μL 完全 DMEM 培养基中。对于每个 96 孔板，将 2.5 x 104 个细胞接种到每个孔中，一个板总共需要 2.5 x 106 个细胞。将96孔板置于37°C的CO2 培养箱中过夜。</li>
	<li>HEK293T（ACE2/TMPRSS2）感染：使用Ha-CoV-2变体颗粒感染HEK293T（ACE2/TMPRSS2）细胞。在感染的早晨，从预先接种的 96 孔板中取出 50 μL DMEM。在37°C下用50μLHa-CoV-2野生型，Delta或Omicron替换培养基18小时。 注意：可以在此处停止该方案，直到感染达到孵育18小时，并且板已准备好通过荧光素酶测定进行分析。感染的成功取决于由感染细胞中表达的荧光素酶报告基因产生的荧光素酶测定，因此产生的信号越多，Ha-CoV-2变体的感染就越成功。</li>
	<li>荧光素酶测定：孵育 18 小时后，将 7.5 μL 细胞裂解缓冲液直接加入每个孔中，并通过轨道振荡混合 2 分钟。在室温下将裂解细胞在裂解缓冲液中裂解至少5分钟。</li>
	<li>通过将D-荧光素溶液与萤火虫荧光素酶测定溶液以1：50的比例混合来制备萤火虫荧光素酶测定溶液。对于整个 96 孔板，将 3 mL 荧光素酶底物溶液与 60 μL D-荧光素溶液与 2940 μL Firefly 荧光素酶缓冲液混合。</li>
	<li>向细胞裂解物中加入25μL萤火虫荧光素酶测定溶液，并通过轨道振荡1分钟混合板。使用商业荧光素酶</li>
</ol>3. Ha-CoV-2 的 RNA 提取和定量逆转录酶 PCR （RT-qPCR）
<ol><li>病毒 RNA 提取：按照制造商的说明，使用商业病毒 RNA 提取试剂盒从 Ha-CoV-2 野生型和 Ha-CoV-2 Delta 和 Omicron 变体颗粒中提取病毒 RNA。将提取的病毒RNA储存在-80°C或立即用于RT-qPCR。</li>
	<li>RT-qPCR：使用一步法预混液对病毒RNA进行RT-qPCR。在商用PCR机中进行反应。请注意，扩增的靶标是 Ha-CoV-2 的基因组 RNA。使用 Ha-CoV-2 载体 DNA 作为标准品，用于创建标准曲线并计算每个变体的 RNA 拷贝数。</li>
</ol>4.中和抗体测定
<ol><li>接种 HEK293T （ACE2/TMPRSS2） 细胞：在测定前一天，将 96 孔板中的 HEK293T （ACE2/TMPRSS2） 细胞接种在 50 μL 完整的 DMEM 培养基中。HEK293T（ACE2/TMPRSS2）电池是商业购买的。</li>
	<li>对于细胞计数，从含有 HEK293T（ACE2/TMPRSS2） 细胞的 T75 烧瓶中获取 20 μL 细胞，并与 20 μL 台盼蓝溶液混合。将 20 μL 该混合物加入细胞计数室并计数每 mL 的细胞数。要接种 96 孔板进行感染，每孔使用 2.5 x 104 个细胞，总共需要 2.5 x 106 个细胞。将96孔板置于37°C的CO2 培养箱中过夜。</li>
	<li>中和抗体测定：在无菌聚丙烯 96 孔板中，制备标准 27BV 中和抗体和 Ha-CoV-2 混合物。向板中加入 8 μL 27BV （45 mg/mL），并用 6 μL 无血清 DMEM 连续稀释抗体。 注意：请务必在孔转移之间交换移液器吸头，并确保抗体和无血清培养基充分混合以产生准确的结果。</li>
	<li>向连续稀释的抗体中加入 54 μL Ha-CoV-2 颗粒，并将病毒和抗体混合。在37°C下以5%CO2连续稀释的27BV预孵育Ha-CoV-2颗粒1小时。孵育 1 小时后，将 50 μL 抗体和 Ha-CoV-2 混合物施加到含有前一天接种的 HEK293T（ACE2/TMPRSS2） 细胞（每孔 2.5 x 104 个细胞）的 96 孔板上。</li>
	<li>对于对照，至少留出三个仅含有 HEK293T（ACE2/TMPRSS2） 细胞的孔。向这些孔中加入 50 μL 完全培养基，作为荧光素酶测定读数的背景信号的非感染孔。 注意：可以在此处停止该方案，直到感染在37°C下达到孵育18小时，然后准备通过荧光素酶测定分析板。</li>
	<li>荧光素酶测定：孵育18小时后，将7.5μL裂解缓冲液直接加入每个孔中，并通过轨道振荡混合2分钟。在室温下将裂解细胞在裂解缓冲液中裂解至少5分钟。</li>
	<li>通过将D-荧光素溶液与萤火虫荧光素酶测定溶液以1：50的比例混合来制备萤火虫荧光素酶测定溶液。对于整个 96 孔板，将 3 mL 荧光素酶底物溶液与 60 μL D-荧光素溶液与 2940 μL 萤火虫荧光素酶缓冲液混合。</li>
	<li>向细胞裂解物中加入25μL萤火虫荧光素酶测定溶液，并通过轨道振荡1分钟混合板。使用商业荧光素酶</li>
</ol>5. 量化和统计分析
<ol><li>数据收集：如图所示，一式三份进行感染和荧光素酶测定（图1）。使用荧光素酶测定读数定量荧光素酶表达。平均值是三个荧光素酶测定读数的平均值。从该平均值中减去来自非感染孔的背景信号读数。标准偏差 （SD） 由荧光素酶测定读数的平均值确定。</li>
	<li>数据分析：绘制抗体中和活性图，并使用商业绘图软件计算ID50 （50%抑制剂量）值。ID50 值定义为抗体抑制稀释度，在该稀释度下，HEK293T（ACE2/TMPRSS2） 细胞感染减少 50%（基于荧光素酶测定读数）。</li>
</ol>

定量 SARS-CoV-2 变体中疫苗诱导的中和抗体

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Weekly epidemiological update on COVID-19. 143, 1 (2023).</li><li>Dhama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+emerging+Omicron+variant+of+SARS-CoV-2:+Impacts,+challenges+and+strategies.">Global emerging Omicron variant of SARS-CoV-2: Impacts, challenges and strategies.</a> Journal of Infection and Public Health. 16 (1), 4-14 (2023).</li><li>Dhama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+Disease+2019-COVID-19.">Coronavirus Disease 2019-COVID-19.</a> Clinical Microbiology Reviews. 33 (4), e00028-e00020 (2020).</li><li>El-Shabasy, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+waves+changes,+new+variant+strains,+and+vaccination+effect+against+COVID-19+pandemic.">Three waves changes, new variant strains, and vaccination effect against COVID-19 pandemic.</a> International Journal of Biological Macromolecules. 204, 161-168 (2022).</li><li>CDC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Interim laboratory biosafety guidelines for handling and processing specimens associated with coronavirus disease 2019 (COVID-19). , (2021).</li><li>Kaufer, A., Theis, T., Lau, K., Gray, J., Rawlinson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+biosafety+measures+involving+SARS-CoV-2+and+the+classification+as+a+Risk+Group+3+biological+agent.">Laboratory biosafety measures involving SARS-CoV-2 and the classification as a Risk Group 3 biological agent.</a> Pathology. 52 (7), 790-795 (2020).</li><li>Vanderheiden, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Rapid+Focus+Reduction+Neutralization+Test+Assay+for+Measuring+SARS-CoV-2+Neutralizing+Antibodies.">Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies.</a> Current Protocols in Immunology. 131 (1), e116 (2020).</li><li>Dieterle, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Replication-Competent+Vesicular+Stomatitis+Virus+for+Studies+of+SARS-CoV-2+Spike-Mediated+Cell+Entry+and+Its+Inhibition.">A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.</a> Cell Host &amp; Microbe. 28 (3), 486-496 (2020).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+SARS-CoV-2+neutralizing+antibody+by+a+pseudotyped+virus-based+assay.">Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.</a> Nature Protocols. 15 (11), 3699-3715 (2020).</li><li>Nie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+validation+of+a+pseudovirus+neutralization+assay+for+SARS-CoV-2.">Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.</a> Emerging Microbes &amp; Infections. 9 (1), 680-686 (2020).</li><li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+effectiveness+of+pseudotyped+SARS-CoV-2+system+as+determined+by+neutralizing+efficiency+and+entry+inhibition+test+in+vitro.">Development and effectiveness of pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro.</a> Biosafety and Health. 2 (4), 226-231 (2020).</li><li>Hetrick, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+hybrid+alphavirus-SARS-CoV-2+pseudovirion+for+rapid+quantification+of+neutralization+antibodies+and+antiviral+drugs.">Development of a hybrid alphavirus-SARS-CoV-2 pseudovirion for rapid quantification of neutralization antibodies and antiviral drugs.</a> Cell reports methods. 2 (3), 100181 (2022).</li><li>Jackson, C., Farzan, M., Chen, B., Choe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+SARS-CoV-2+entry+into+cells.">Mechanisms of SARS-CoV-2 entry into cells.</a> Nature Reviews. Molecular Cell Biology. 23 (1), 3-20 (2022).</li><li>Jackson, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+mRNA+Vaccine+against+SARS-CoV-2+-+Preliminary+Report.">An mRNA Vaccine against SARS-CoV-2 - Preliminary Report.</a> The New England Journal of Medicine. 383 (20), 1920-1931 (2020).</li><li>Polack, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Efficacy+of+the+BNT162b2+mRNA+Covid-19+Vaccine.">Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine.</a> The New England Journal of Medicine. 383 (27), 2603-2615 (2020).</li><li>Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+evolution+of+coronavirus+spike+proteins.">Structure, function, and evolution of coronavirus spike proteins.</a> Annual Review of Virology. 3 (1), 237-261 (2016).</li><li>Donofrio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+SARS-CoV-2+Pseudovirus+Neutralization+Assay.">A Simplified SARS-CoV-2 Pseudovirus Neutralization Assay.</a> Vaccines. 9 (4), 389 (2021).</li><li>Crawford, K. H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+and+Reagents+for+Pseudotyping+Lentiviral+Particles+with+SARS-CoV-2+Spike+Protein+for+Neutralization+Assays.">Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.</a> Viruses. 12 (5), 513 (2020).</li><li>Zettl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Quantification+of+SARS-CoV-2-Neutralizing+Antibodies+Using+Propagation-Defective+Vesicular+Stomatitis+Virus+Pseudotypes.">Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes.</a> Vaccines. 8 (3), 386 (2020).</li><li>Li, Q., Liu, Q., Huang, W., Li, X., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+on+the+development+of+pseudoviruses+for+enveloped+viruses.">Current status on the development of pseudoviruses for enveloped viruses.</a> Reviews in Medical Virology. 28 (1), e1963 (2018).</li><li>Lundin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+firefly+luciferase+reaction+for+analytical+purposes.">Optimization of the firefly luciferase reaction for analytical purposes.</a> Advances in Biochemical Engineering/Biotechnology. 145, 31-62 (2014).</li><li>Deshpande, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+responses+to+SARS-CoV-2+in+COVID-19+patients.">Neutralizing antibody responses to SARS-CoV-2 in COVID-19 patients.</a> The Indian Journal of Medical Research. 152 (1 &#38; 2), 82-87 (2020).</li><li>Chen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+in+methods+for+detecting+neutralizing+antibodies+against+SARS-CoV-2.">Research progress in methods for detecting neutralizing antibodies against SARS-CoV-2.</a> Analytical Biochemistry. 673, 115199 (2023).</li><li>Manischewitz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+vaccinia-neutralization+assay+based+on+reporter-gene+expression.">Development of a novel vaccinia-neutralization assay based on reporter-gene expression.</a> The Journal of Infectious Diseases. 188 (3), 440-448 (2003).</li><li>Chen, J., Wang, R., Gilby, N., Wei, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omicron+Variant+(B.1.1.529):+Infectivity,+Vaccine+Breakthrough,+and+Antibody+Resistance.">Omicron Variant (B.1.1.529): Infectivity, Vaccine Breakthrough, and Antibody Resistance.</a> Journal of Chemical Information and Modeling. 62 (2), 412-422 (2022).</li><li>Saxena, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+novel+SARS-CoV-2+Omicron+(B.1.1.529)+variant+of+concern+and+its+global+perspective.">Characterization of the novel SARS-CoV-2 Omicron (B.1.1.529) variant of concern and its global perspective.</a> Journal of Medical Virology. 94 (4), 1738-1744 (2022).</li></ol>

乔治梅森大学已申请专利，并授权给Virongy Biosciences Inc.进行产品开发。Y.W.是Virongy Biosciences的创始人和顾问委员会成员。B. H 目前是 Virongy Biosciences 的首席执行官兼首席科学官。作者没有其他利益冲突。

Ha-CoV-2 平台提供了快速、稳健和简单的工作流程来量化病毒变异和中和抗体。但是，有几个关键步骤需要注意。Ha-CoV-2 假病毒的生产应使用具有高活力的HEK293T细胞进行。可以使用来自 Ha-CoV-2 基因组的 GFP 报告基因在转染后 24 小时监测共转染效率。Ha-CoV-2 基因组可包含两个报告基因（GFP 和 Luc），GFP 可在共转染期间和 Ha-CoV-2 感染靶细胞后表达12。感染的GFP+细胞通常处于低百分比（1%至5%），但每个感染细胞都表达强烈的GFP信号（图3）。与定量整个感染细胞群的 Luc 报告基因相比，这种低 GFP 百分比可能会限制 GFP 作为定量抗体中和的可靠读数的使用。
进行中和测定时，必须在孔转移之间更换移液器吸头，并确保抗体和无血清培养基充分混合以产生准确的结果。此外，在进行荧光素酶测定方案时，细胞必须完全裂解至少3分钟，以确保细胞完全裂解和荧光素酶的释放。这将确保测定的准确性。此外，一旦将萤火虫荧光素酶测定溶液添加到光学白壁 96 孔板中，必须在 10 分钟内分析板，因为初始光发射很高，但随着 ATP 耗尽，随着时间的推移会降低21。
随着越来越多的 SARS-CoV-2 变异株不断进化，对 Ha-CoV-2 等平台的需求越来越大，以快速筛查变异株的传染性和对疫苗诱导的中和抗体的变异株敏感性。与现有的基于假病毒的中和检测相比，Ha-CoV-2 平台具有更快的速度、更高的信噪比和简单的实验方案 8,9,10,11。Ha-CoV-2平台还具有可用于BSL-2实验室的优势，并且不需要使用BSL-3设施。这使得 SARS-CoV-2 可以在共同的研究和临床实验室中进行研究。此外，与其他系统相比，Ha-CoV-2平台可快速产生结果。例如，针对传染性 SARS-CoV-2 病毒的中和抗体的研究通常使用斑块减少中和试验 （PRINT）22。尽管 PRINT 可产生可靠的结果，但对斑块形成单位 （PFU） 的手动计数速度很慢，需要 3-5 天才能获得结果23,24。其它假型系统，如慢病毒-假病毒需要24-72小时才能产生可检测的报告基因信号12。相比之下，Ha-CoV-2 中和测定可以在 18 小时内产生结果。Ha-CoV-2为快速筛查和定量病毒变异株以及用于大流行监测的中和抗体提供了一种方便的工具。
随着更多需要关注的变异株 （VOC） 不断出现，监测 SARS-CoV-2 的传染性至关重要。Ha-CoV-2 具有快速确定 VOC 传染性的优势。以前的研究使用基于人工智能 （AI） 的建模来定量分析 Omicron 亚变体和其他 SARS-CoV-2 变体（例如 Delta变体 25）的传染性。这些研究表明，奥密克戎变异株比原始病毒更具传染性，并且更有可能逃避中和抗体25。在这些研究中，使用Ha-CoV-2，观察到类似的表型。此外，在抗体中和试验中，奥密克戎变异株被27BV中和的可能性是武汉和德尔塔毒株的十倍。这些结果也与报道的奥密克戎变异株的较高传播性一致，奥密克戎变异株的受体结合域（RBD）上至少有15个突变，可能增强了病毒与ACE2受体的结合亲和力，从而实现了更高的传播性和更大的免疫逃逸26。

截至 2023 年 5 月，目前已有超过 7.66 亿例 COVID-19 病例1.尽管在全球范围内开展了疫苗接种运动，但 SARS-CoV-2 仍在不断传播并感染人们，这主要是由于 Delta （B.1.617.2） 和 Omicron （B.1.1.529） 等新变种的出现推动了新一波感染浪潮 2,3,4。鉴于 SARS-CoV-2 在不断发展，开发能够准确测量新变异株的传染性以及疫苗诱导的针对这些变异株的中和抗体的有效性的快速检测非常重要。这些检测对于大流行监测和确定疫苗及其变异株特异性加强针的功效至关重要。
由于 SARS-CoV-2 具有高度传染性，美国疾病控制与预防中心 （CDC） 要求在生物安全级别 （BSL） 3 设施中对 SARS-CoV-2 及其变体进行研究 5,6。BSL-3 的这一要求限制了在普通研究和临床实验室中使用活病毒来量化病毒变体及其中和抗体的传染性。此外，传统的 SARS-CoV-2 中和检测，例如使用具有复制能力的活病毒的基于斑块或细胞病变效应的检测，非常耗时且需要较长的潜伏期7。已经开发了几种刺突 （S） 蛋白假型 SARS-CoV-2 假病毒来量化中和抗体8、9、10、11、12 的有效性。在 SARS-CoV-2 中，S 蛋白是介导病毒进入的主要蛋白质 13，也是 SARS-CoV-2 疫苗中使用的主要抗原 9,10,14,15,16。S 蛋白假型病毒粒子，例如水泡性口炎病毒 （VSV-G） 或慢病毒的病毒粒子，已用于中和抗体的定量 17,18,19。然而，基于慢病毒的假病毒通常需要 2 到 3 天的感染才能量化报告基因信号。基于 VSV 的假病毒系统通常包含残留的 VSV 病毒，这可能导致高假阳性结果率，通常需要 24 小时的感染20。
Hetrick 等人最近开发了一种新型 SARS-CoV-2 假病毒系统，即混合甲病毒-SARS-CoV-2 假病毒 （Ha-CoV-2）12。Ha-CoV-2 为在普通 BSL-2 实验室中快速定量病毒传染性和病毒对中和抗体的敏感性提供了一种新工具。在结构上，Ha-CoV-2 类似于 SARS-CoV-2 病毒粒子，由 SARS-CoV-2 结构蛋白组成，包括 S 蛋白 （S）、膜 （M）、核衣壳 （N） 和包膜 （E），并且没有来自其他病毒的结构蛋白。此外，Ha-CoV-2 颗粒含有来自甲病毒的快速表达 RNA 基因组，可在细胞中快速表达报告基因。Ha- CoV-2 已被证明可以快速测量接种疫苗和康复期个体血清中抗体的中和活性12。正如 Hetrick 等人所证明的那样，在时程测定中与基于慢病毒的 SARS-CoV-2 假病毒相比，Ha-CoV-2 早在感染后 2-4 小时表达 Luc 报告基因，而慢病毒假病毒在 24 小时12 后表达 Luc。此外，通过使用标准单克隆中和抗体 27BV（见 补充图 1）12，进一步证明了 Ha-CoV-2 变体在定量中和抗体方面的潜在应用。这项工作详细介绍了使用 Ha-CoV-2 平台快速量化 SARS-CoV-2 变体的传染性，以 Delta （B.1.617.2） 和 Omicron （B.1.1.529） 变体为例。此外，通过使用标准单克隆中和抗体 27BV12，进一步证明了 Ha-CoV-2 变体在定量中和抗体方面的潜在应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>27VB1 20 &#181;g SARS-CoV-2 Standard Neutralizing Antibody</td>
			<td>Virongy Biosciences</td>
			<td>27VBI-01</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL - US Origin FBS</td>
			<td>Neuromics</td>
			<td>FBS001</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Mixing Plate: Olympus 96-Well PCR Plate, Non-Skirted UltraThin Wall, Natural, 25 Plates/Unit&#160;</td>
			<td>Genesee Scientific</td>
			<td>Cat# 24-300&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra 6R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>2043-30-1158</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (1x)&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>GenClone 25-209, TC Treated Flasks, 250ml, Vent Growth Area: 75.0cm2, 5 per Sleeve, 100 Flasks/Unit</td>
			<td>Genesee Scientfic</td>
			<td>25-209</td>
			<td></td>
		</tr>
		<tr>
			<td>GlowMax Discover Microplate reader</td>
			<td>Promega&#160;</td>
			<td>GM3000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ha-CoV-2 E Vector&#160;</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2_E</td>
			<td></td>
		</tr>
		<tr>
			<td>Ha-CoV-2 M Vector&#160;</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2_M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ha-CoV-2 N Vector&#160;</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2_N</td>
			<td></td>
		</tr>
		<tr>
			<td>Ha-CoV-2 WT S Vector</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2_WT S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hek293T cells</td>
			<td>ATCC</td>
			<td>CRL-3214</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumination Firefly Luciferase Enhanced Assay Kit 1000 assays</td>
			<td>Gold Bio&#160;</td>
			<td>I-930-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Infection Plate: 96-Well Tissue Culture Plate, Greiner Bio-One (With Lid, &#956;Clear White Flat Round, Chimney)&#160;</td>
			<td>VWR&#160;</td>
			<td>Cat# 82050-758&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pAlphaPro-Luc-GFP-Pre&#936; (Ha-CoV-2 Genome) Vector&#160;</td>
			<td>In house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEI-based Transfection Reagent</td>
			<td>Virongy Biosciences</td>
			<td>Transfectin</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Glutamine (100X)</td>
			<td>Invitrogen</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine, branched</td>
			<td>Millipore Sigma</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 7 Pro Real-Time PCR System</td>
			<td>ThermoFisher&#160;</td>
			<td>A43163</td>
			<td></td>
		</tr>
		<tr>
			<td>Ready to use (HEK293T)(ACE2/TMPRSS2) Cells</td>
			<td>Virongy Biosciences</td>
			<td>Ready-To-Use-Cells</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 S&#160; Omicron (B.1.1.529) Vector</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2-B.1.1.529</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 S Delta (B.1.617.2)&#160;Vector</td>
			<td>Virongy Biosciences</td>
			<td>pCoV2- B.1.617.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters, PES, 0.22&#181;m</td>
			<td>Genesee Scientfic</td>
			<td>25-244</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Fast Virus 1-Step Master Mix</td>
			<td>ThermoFisher&#160;</td>
			<td>4444432</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>ThermoFisher&#160;</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of ha-cov-2 pseudovirus for rapid quantification of sars-cov-2 variants and neutralizing antibodies

2019 年冠状病毒病大流行 （COVID-19） 凸显了快速检测的必要性，以准确测量新出现的 SARS-CoV-2 变体的传染性以及疫苗诱导的针对病毒变体的中和抗体的有效性。这些检测对于大流行监测和验证疫苗和变异株特异性加强针至关重要。这篇手稿展示了一种新型混合型甲病毒-SARS-CoV-2 假病毒 （Ha-CoV-2） 在快速定量 SARS-CoV-2 变异株感染性和疫苗诱导的病毒变异中和抗体中的应用。 Ha-CoV-2 是一种 SARS-CoV-2 病毒样颗粒，由病毒结构蛋白（S、M、N 和 E）和源自甲病毒塞姆利基森林病毒 （SFV） 的快速表达 RNA 基因组组成。Ha-CoV-2 还含有绿色荧光蛋白 （GFP） 和荧光素酶报告基因，可快速定量病毒感染性。例如，量化了 SARS-CoV-2 Delta （B.1.617.2） 和 Omicron （B.1.1.529） 变体的传染性，并测量了它们对中和抗体 （27VB） 的敏感性。这些例子表明，Ha-CoV-2 作为快速定量 SARS-CoV-2 变体及其对中和抗体敏感性的强大平台具有巨大潜力。

Ha-CoV-2 颗粒使用五种不同的 DNA 载体组装，这些载体在HEK293T细胞中表达 Ha-CoV-2 RNA 基因组和 SARS-CoV-2 的结构蛋白（M、N、E 和 S）。S 蛋白载体因 S 变体而异。来自原始 Ha-CoV-2 武汉菌株（野生型，Wt）的 S 蛋白被用作阳性对照，并将其与其他两个变体的 S 蛋白一起组装：Delta （B.1.617.2） 或 Omicron （B.1.1.529）。所有变体都使用相同的 M、N、E。共转染后 48 小时收集 Ha-CoV-2（Wt） 和变异颗粒，然后用于感染 HEK293T（ACE2/TMPRSS2） 细胞。通过感染后 18 小时荧光素酶的表达来测量感染性。在该系统中，荧光素酶信号的较高表达水平反映了 Ha-CoV-2 对细胞的较高感染。荧光素酶信号通过RT-qPCR对每个变体的基因组RNA拷贝进行归一化。如 图 1 所示，Ha-CoV-2 Omicron 变体产生的信号比原始 Ha-CoV-2（Wt） 高 4 至 10 倍，表明传染性更高。
此外，还量化了 27BV 中和 HaCoV-2（Wt）、Delta 和 Omicron 变体的能力。27BV 是一种针对 SARS-COV-2 S1 蛋白的 RBD 结构域开发的兔单克隆抗体。对于中和测定，在 96 孔板中连续稀释 27BV，与 Ha-CoV-2 预孵育，然后加入 HEK293T（ACE2/TMPRSS2） 靶细胞。结果表明，27BV对所有测试的变体都具有中和活性（图2）。有趣的是，27VB 对 Omicron 的 ID50 的效力比 Ha-CoV-2（WT） 和 Ha-CoV-2（Delta;图2）。这些结果表明，Ha-COV-2平台可以作为一种快速方法，用于量化新变异株中疫苗诱导的中和抗体。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65793/65793fig01.jpg"> 图 1.Ha-CoV-2变体的组装和定量。 （A） Ha-CoV-2和变异颗粒的组装图示。表达 Ha-CoV-2 报告基因基因组和结构蛋白（M、S、N 和 E）的载体在HEK293T细胞中共转染。共转染后 48 小时收获颗粒（用 Biorender.com 创建病毒粒子颗粒和HEK293T细胞的成像）。（B） Ha-CoV-2变异株传染性的量化。使用单个 Ha-CoV-2 （Luc） 变体的基因组 RNA 拷贝对两种变体（Delta 和 Omicron）的相对传染性进行量化和归一化。野生型是 Ha-COV-2 （Wt），用作比较的对照。进行感染和荧光素酶测定 3x. RU，相对单位。显示均值和标准差 （SD）。 <a href="https://www.jove.com/files/ftp_upload/65793/65793fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65793/65793fig02.jpg"> 图2.定量27BV对Ha-CoV-2（Luc）变异株的中和活性 在HEK293T（ACE2/TMPRSS2）细胞感染后18小时分析了27BV的中和活性。ID50 使用相对感染率（荧光素酶活性）与 27BV 浓度计算。 <a href="https://www.jove.com/files/ftp_upload/65793/65793fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65793/65793fig03.jpg"> 图3.Ha-CoV-2（GFP）感染HEK293T（ACE2/TMPRSS2）。 组装Ha-CoV-2（GFP）颗粒，然后用于感染HEK293T（ACE2/TMPRSS2）细胞。使用荧光显微镜观察感染后 48 小时的 GFP 表达。左边是受感染细胞的白场，右边是GFP成像。白色条代表 100 μm。 <a href="https://www.jove.com/files/ftp_upload/65793/65793fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图 1.图形摘要。 Ha-CoV-2假病毒结构和应用。使用 Biorender.com 创建的图像。 <a href="https://www.jove.com/files/ftp_upload/65793/Graphical%20Abstract_RE.pdf" target="_blank">请点击此处下载此文件。</a>
补充文件 1.蛋白质序列。 SARS-CoV-2 S、M、N 和 E 蛋白的序列列表。S 蛋白序列还包括 SARS-CoV-2 变体 Omicron （B.1.1.529） 和 Delta （B.1.617.2）。 <a href="https://www.jove.com/files/ftp_upload/65793/Protein%20Sequences_joVE_RE.docx" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies at JoVE.com

作者聚焦：用于评估 SARS-CoV-2 研究中 Omicron 亚变体和中和抗体的假型病毒系统

该协议描述了新型混合甲病毒-SARS-CoV-2假病毒（Ha-CoV-2）的应用，作为快速定量SARS-CoV-2变体的传染性及其对中和抗体的敏感性的平台。

Ha-CoV-2假病毒在SARS-CoV-2变异株和中和抗体快速定量中的应用

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...

我们在该协议中建立了两个主要发现。首先，我们的 Ha-CoV-2 平台表明，新出现的 Omicron 亚变体比原始武汉毒株更具传染性。此外，奥密克戎亚变体对先前毒株诱导的中和抗体更具抵抗力。SARS CoV-2 研究需要 BSL-3 设施，这使得普通实验室难以监测 SARS-CoV-2 变体并量化中和抗体。我们的 Ha-CoV-2 系统是一种假型病毒，可用于普通实验室，并提供了一种更快的方法来量化病毒变体及其对中和抗体的敏感性。Ha-CoV-2 是一种病毒样颗粒，具有 SARS-CoV-2 的所有结构蛋白，类似于 SARS-CoV-2。此外，Ha-CoV-2 使用 α 病毒载体进行稳健和快速的基因表达，产生快速结果和高信噪比，从而实现出色的可重复性。

application-ha-cov-2-pseudovirus-for-rapid-quantification-sars-cov-2

Research

JoVE Journal

Immunology and Infection

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

1.0K 次观看.  George Mason University. 我们在该协议中建立了两个主要发现。首先，我们的 Ha-CoV-2 平台表明，新出现的 Omicron 亚变体比原始武汉毒株更具传染性。此外，奥密克戎亚变体对先前毒株诱导的中和抗体更具抵抗力。SARS CoV-2 研究需要 BSL-3 设施，这使得普通实验室难以监测 SARS-CoV-2 变体并量化中和抗体。我们的 Ha-CoV-2 系统是一种假型病毒，可用于普通实验室，并提供了一种更快的方法来量化病毒?...

视频: 作者聚焦：用于评估 SARS-CoV-2 研究中 Omicron 亚变体和中和抗体的假型病毒系统

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

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 ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

双光子激光显微镜在细胞膜修复中的应用

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

抗原脂质体用于生成特定于疾病的抗体

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

基于多路复用珠子的流量分析系统的快速、多路双记者 IgG 和 Igm SARS-CoV-2 中和检测

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...