PDF(Pubmed)
Abstract：
Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine.
OBJECTIVE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important \"gold standard\" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.
OBJECTIVE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important \"gold standard\" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.
摘要：
血清中和抗体滴度是疫苗免疫原性的关键指标，可用于确定研究人群中黄病毒的血清阳性率。有效的登革热病毒（DENV）疫苗必须同时提供针对四种抗原血清型的病毒的保护。现有的黄病毒中和试验，包括常用的斑块/病灶减少中和滴度（PRNT/FRNT）测定，需要对每种病毒进行单独检测，血清型,并且容易成为大型流行病学研究或疫苗试验的劳动密集型和耗时的工作。这里,我们描述了一种用于DENV的多重报告病毒颗粒中和滴度(TetraPlexRVPNT)检测方法,该方法允许同时定量测量单个低容量临床样品中针对所有4种DENV血清型的抗体介导的感染中和,并通过流式细胞术进行分析.比较研究证实,通过TetraPlexRVPNT测定测量的抗体的中和滴度类似于对每种病毒株分别进行的FRNT/PRNT测定方法。使用这种高通量方法能够在DENV流行人群和疫苗接受者中进行仔细的血清学研究，以支持开发安全有效的四价DENV疫苗。
目的：作为登革热疾病的保护介质和先前感染的血清学指标，针对DENV的中和抗体的检测和定量是重要的“金标准”工具。然而,传统的中和抗体测定的执行通常是麻烦的,并且需要对每种病毒或血清型重复应用。这里描述的优化的RVPNT测定是高通量的，很容易在多种血清型之间多路复用，和靶报道病毒颗粒,其可以对于所有四种DENV血清型稳健地产生。这种转化性RVPNT测定的使用将支持中和抗体数据集的扩展,以回答通常受到更繁琐的中和抗体测定和对更大量的测试血清的需要限制的研究和公共卫生问题。
目的：作为登革热疾病的保护介质和先前感染的血清学指标，针对DENV的中和抗体的检测和定量是重要的“金标准”工具。然而,传统的中和抗体测定的执行通常是麻烦的,并且需要对每种病毒或血清型重复应用。这里描述的优化的RVPNT测定是高通量的，很容易在多种血清型之间多路复用，和靶报道病毒颗粒,其可以对于所有四种DENV血清型稳健地产生。这种转化性RVPNT测定的使用将支持中和抗体数据集的扩展,以回答通常受到更繁琐的中和抗体测定和对更大量的测试血清的需要限制的研究和公共卫生问题。
