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生存和扩张 几种传播虫媒病毒的蚊子和蜱虫。在城市中茁壮成长的伊蚊 人类创造的环境是几种黄病毒[登革病毒（DENV）]流行的原因 血清型1、2、3、4;寨卡病毒（ZIKV）;黄热病病毒（YFV）]和甲病毒[基孔肯雅病毒（CHIKV） 和马亚罗病毒（MAYV）]。虫媒病毒的诊断和监测很困难，因为 大多数感染者没有症状或发展为轻度未分化发热性疾病。血清学测定 已经开发了用于检测最近或过去的虫媒病毒感染，但这些测定的实用性 受到相关病毒之间抗体交叉反应性的严重限制。例如，当ZIKV出现在许多 在美洲地区，超过80%的人口对登革热免疫，目前的血清学 由于ZIKV的检测，很难（如果不是不可能的话）在一定的时间内识别受感染的个体或监测ZIKV的传播。 人口水平，同样地，现在在经历过强烈ZIKV的地区检测新的DENV感染 流行病我们过去10年的研究表明，暴露于黄病毒感染的人可靠地 开发针对每种黄病毒独特表位的抗体以及交叉反应抗体。使用我们 关于免疫显性病毒类型特异性表位的位置的发现，我们已经产生了新的 重组抗原，并证明了它们的实用性的虫媒病毒的类型特异性诊断。下 本提案的具体目标1，我们将在这些发现的基础上开发一种样品保留， 基于微球珠的多重检测，用于最近或过去的类型特异性和灵敏性检测 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 白纹伊蚊，因为这些病毒共享相似的生态，并在同一个人 人口。在第二阶段，我们将扩大检测的覆盖范围，以检测其他 由其他蚊子和蜱传播的虫媒病毒。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。虽然疫苗开发者依赖于中和 抗体作为保护的相关物，最近的临床试验结果表明， 单独与疫苗安全性和有效性相关性较差。我们已经确定了黄病毒的类型和表位特异性 抗体应答比中和抗体更能预测疫苗的安全性和有效性。下 本提案的具体目标2，我们将开发一种基于样品保留微球的检测方法， 表位特异性疫苗诱导的抗体应答的检测， 对DENV血清型和ZIKV中的每一种的免疫力。技术进步和由此产生的产品 建议将提高我们在个人和人口层面有效监测虫媒病毒感染的能力 也支持虫媒病毒疫苗的研发。

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.