Multiplex serological assays to support arbovirus diagnosis, surveillance and vaccines

摘要 在全球范围内，持续的生态、环境和人口变化有利于人类的生存和扩张 传播虫媒病毒的几种蚊子和扁虱。在城市繁衍生息的伊蚊物种 人类创造的环境是几种黄病毒[登革热病毒(DENV)]流行的原因 血清型1、2、3、4型；寨卡病毒(ZIKV)；黄热病病毒(YFV)；甲型病毒[基孔肯雅病毒(CHIKV) 和马亚罗病毒(MAYV)]。虫媒病毒的实验室诊断和监测很困难，因为 大多数感染者没有症状或发展为轻微的未分化发热性疾病。血清学检测 已经被开发用于检测最近或过去的虫媒病毒感染，但这些分析的实用是 严重受限于相关病毒之间的抗体交叉反应。例如，当ZIKV出现在许多 美洲地区，超过80%的人口对登革热免疫，目前的血清学 化验表明，即使不是不可能，也很难识别受感染的个人或监测寨卡病毒的传播。 人口水平，同样现在在寨卡病毒高发地区检测新的DENV感染 流行病。我们过去10年的研究表明，接触黄病毒感染的人可靠地 开发针对每个黄病毒独有的表位的抗体以及交叉反应抗体。使用我们的 关于免疫优势病毒类型特异性表位位置的发现，我们产生了新的 重组抗原，并证明了它们在虫媒病毒类型特异性诊断中的有效性。在……下面 这项建议的具体目标1，我们将在这些发现的基础上开发一种样本-Sparing， 微球微球为基础的多重检测方法，用于最近或过去的类型特异性和敏感性检测 虫媒病毒感染。我们的初步研究将集中在埃及伊蚊和伊蚊传播的8种虫媒病毒上 白纹伊蚊，因为这些病毒共享相似的生态，并在同一人体内共同传播 人口。在第二阶段，我们将扩大化验的覆盖范围，以检测其他 由其他蚊子物种和扁虱传播的虫媒病毒。几种四价DENV和ZIKV疫苗是 目前正在人体临床试验中进行评估。而疫苗研发人员则依赖于中和 抗体作为一种相关的保护，最近的临床试验结果表明，中和抗体 仅此一项与疫苗安全性和有效性的相关性很差。我们已经确定了黄病毒的类型和表位特异性 抗体反应比中和抗体更能预测疫苗的安全性和有效性。在……下面 这项提案的具体目标2，我们将开发一种基于样品节省微球的分析方法 检测与保护性相关的表位特异性疫苗诱导的抗体反应 对每一种DENV血清型和寨卡病毒免疫。技术进步和由此产生的产品 建议将加强我们在个人和人群层面有效监测虫媒病毒感染的能力 并支持虫媒病毒疫苗的开发。

Abstract At a global level, ongoing ecological, environmental and demographic changes favor the survival and expansion of several mosquito and tick species that transmit arboviruses. Aedes mosquito species that thrive in urban environments created by humans are responsible for epidemics of several flaviviruses [dengue virus (DENV) serotypes 1, 2, 3, 4; Zika virus (ZIKV); yellow fever virus (YFV)] and alphaviruses [chikungunya virus (CHIKV) and Mayaro virus (MAYV)]. Laboratory-based diagnosis and surveillance for arboviruses is difficult because most infected individuals are asymptomatic or develop a mild undifferentiated febrile illness. Serological assays have been developed for the detection of recent or past arboviral infections, but the utility of these assays is severely limited by antibody cross reactivity between related viruses. For example, when ZIKV emerged in many regions of the Americas where greater than 80% of the population was dengue-immune, with current serological assays, it was difficult, if not impossible, to identify infected individuals or monitor the spread of ZIKV at a population level, and likewise to now detect new DENV infections in areas that experienced intense ZIKV epidemics. Our studies over the past 10 years demonstrate that people exposed to flavivirus infections reliably develop antibodies to epitopes that are unique to each flavivirus as well as cross-reactive antibodies. Using our discoveries about the location of immunodominant virus type-specific epitopes, we have produced novel recombinant antigens and demonstrated their utility for the type-specific diagnosis of arboviruses. Under Specific Aim 1 of this proposal, we will build on these discoveries to develop a sample-sparing, microsphere bead-based multiplex assay for the type-specific and sensitive detection of recent or past arbovirus infections. Our initial studies will focus on 8 arboviruses transmitted by Aedes aegypti and Aedes albopictus mosquitos because these viruses share a similar ecology and co-circulate in the same human populations. At a second stage, we will expand the coverage of the assay to detect infections with other arboviruses transmitted by other mosquito species and ticks. Several tetravalent DENV and ZIKV vaccines are currently being evaluated in human clinical trials. While vaccine developers have relied on neutralizing antibodies as a correlate of protection, recent results from clinical trials demonstrate that neutralizing antibodies alone are a poor correlate of vaccine safety and efficacy. We have identified flavivirus type- and epitope-specific antibody responses that are better predictors than neutralizing antibodies of vaccine safety and efficacy. Under Specific aim 2 of this proposal, we will develop a sample-sparing microsphere-based assay for the detection of epitope-specific vaccine-induced antibody responses that are correlated with protective immunity to each of the DENV serotypes and ZIKV. The technological advances and products from this proposal will enhance our ability to efficiently monitor arbovirus infections at the individual and population levels and also support the development of arbovirus vaccines.