Analysis of the neutralizing antibody response following flavivirus infection

登革病毒(DENV)是黄病毒属中一种由蚊子传播的成员，由于其广泛的分布和在人类中引起严重疾病的能力，对公共卫生具有全球影响。每年有5000-1亿人感染登革热病毒，其临床表现从自限性急性发热病(登革热)到以血浆渗漏和休克为特征的潜在致命综合征(登革出血热；DHF)。四种相关的DENV血清型在自然界中循环，每一种都能够引起DENV相关疾病的全部谱系。前瞻性临床研究清楚地表明，连续感染两种DENV血清型与更严重的病程有关。在过去20年中，报告的DHF病例数量急剧增加，现在每年超过25万例。因此，迫切需要开发一种针对所有四种血清型DENV的安全有效的疫苗。 中和抗体在预防黄病毒感染方面发挥着重要作用。抗体已经被映射到E蛋白(DI-DIII)的所有三个结构域，这些结构域显示出不同程度的中和效力，并通过多种效应机制提供保护。获得中和抗体是疫苗研制的主要目标。使这些努力复杂化的是，疫苗要求同时对四种不同的病毒产生保护，这些病毒虽然在抗原性上相关，但只共享一些被认为有助于病毒中和的抗体结合决定因素。因此，针对一种血清类型的DENV产生的抗体可能与另一种血清类型的病毒粒子反应，但通常亲和力和功能效力降低。 矛盾的是，抗体也可能在增强病毒感染和加重疾病方面发挥作用。抗体依赖的感染增强(ADE)描述了在抗体或免疫血清的亚中和浓度存在的情况下，携带Fc受体的细胞的感染显著增加。ADE和DENV感染的临床结果之间最直接的联系来自对婴儿在出生第一年观察到的原发感染后异常大量的DHF病例的调查。在出生时，DENV特异性被动获得性抗体在相对较高的浓度下存在，并在体外显示出中和活性。然而，随着儿童年龄的增长，母体获得的抗体继续降解到不再具有保护作用的水平，不能中和病毒，并在体外增加病毒感染。在体外，婴儿的抗体效价下降到支持ADE的水平，与出生第一年的初级DENV感染后发生DHF的风险平行。在更广泛的背景下，一次感染一种血清型DENV引起的抗体可能与二次感染期间引入的相关病毒结合，亲和力降低，导致病毒粒子与化学计量比结合，该化学计量比不允许病毒中和，但可以支持ADE。发展一种免疫反应，引起针对疫苗中存在的所有四种血清型病毒的中和抗体的保护性水平，是降低ADE风险的关键因素。 保护性四价反应的发展因以下可能性而变得复杂：减毒活疫苗的四种成分在受种者中并不都具有同等的免疫原性。据报道，在这方面，DENV毒株之间的干扰和传染性水平参差不齐。了解四价疫苗每个成分的免疫原性是疫苗开发和确定适当的保护相关性的一个重要方面，特别是因为目前尚不清楚需要多少血清型特异性反应才能保护所有四种血清型的DENV。然而，由于存在结合疫苗不同成分共享的交叉反应决定因素的抗体，剖析四价疫苗每种成分的具体贡献在技术上具有挑战性。目前还没有E蛋白上重要的类型特异性和基团反应性表位的详细图谱。改进的方法学区分和量化四价疫苗每一组分的功能贡献，将有助于改进疫苗免疫原性的估计，更精确地关联保护，并成为研究疫苗成败的强大调查工具。 为此，我们建议开发一种新的方法，在高度交叉反应的四价多克隆反应的背景下，测量血清型特异性抗体的功能贡献。一组含有破坏抗体结合决定簇的突变的报告病毒颗粒(RVP)将被用来识别在DENV疫苗接种者血清中出现的类型特异性抗体或交叉反应抗体的重要功能表位。迭代筛选过程将指导选择对交叉反应抗体中和不再敏感或对类型特异性反应不再敏感的RVP。具有这些特征的RVP提供了两种互补的和特定的方法来检测多克隆血清中的类型特异性反应，如下所述。然后，这些工具将被用来表征组成疫苗接种或自然感染人类的中和抗体反应的表位。

Dengue virus (DENV) is a mosquito-borne member of the Flavivirus genus that has a global impact on public heath due to its widespread distribution and the ability to cause severe disease in humans. Each year, 50-100 million individuals are infected by DENV, with clinical manifestations ranging from a self-limiting acute febrile illness (dengue fever) to a potentially fatal syndrome characterized by plasma leakage and shock (dengue hemorrhagic fever; DHF). Four related serotypes of DENV circulate in nature, each capable of causing the full spectrum of DENV-related disease. Prospective clinical studies clearly demonstrate that sequential infection with two DENV serotypes is associated with a more severe disease course. The number of DHF cases reported has increased dramatically during the past twenty years, and now exceeds 250,000 cases annually. Thus, there is an urgent need for the development of a safe and effective vaccine for all four serotypes of DENV. Neutralizing antibodies play an important role in protection against flavivirus infection. Antibodies have been mapped to all three structural domains of the E protein (DI-DIII) that exhibit varying degrees of neutralization potency and confer protection by multiple effector mechanisms. Eliciting neutralizing antibody is a major goal of vaccine development. Complicating these efforts is a requirement for vaccines to simultaneously elicit protection against four different viruses that while antigenically related, share only some of the antibody-binding determinants thought to contribute to virus neutralization. Thus, antibodies raised against one serotype of DENV may react with virions of another serotype, but often with reduced affinity and functional potency. Paradoxically, antibodies may also play a role in enhancing virus infection and exacerbating disease. Antibody-dependent enhancement of infection (ADE) describes a dramatic increase in infection of Fc-receptor-bearing cells in the presence of sub-neutralizing concentrations of antibody or immune sera. The most direct link between ADE and the clinical outcome of DENV infection comes from investigations of the unusually large number of DHF cases following primary infection observed in infants during the first year of life. At birth, DENV-specific passively acquired antibodies are present at a relatively high concentration and exhibit neutralizing activity in vitro. However, as the child ages, degradation of maternally acquired antibody continues to levels that are no longer protective, do not neutralize virus, and enhance virus infection in vitro. The waning antibody titers of infants to levels that support ADE in vitro parallels the risk of DHF following primary DENV infection during the first year of life. In a broader context, antibodies elicited by primary infection with one serotype of DENV may bind related viruses introduced during secondary infection with reduced avidity, resulting in engagement of the virion with a stoichiometry that does not permit virus neutralization but can support ADE. The development of an immune response that elicits protective levels of neutralizing antibodies against all four serotypes of virus present in the vaccine is a key factor for reducing the risk of ADE. The development of a protective tetravalent response is complicated by the possibility that not all four components of a live attenuated tetravalent vaccine may be equally immunogenic in the vaccinee. Interference and uneven levels of infectivity among DENV strains in this context has been reported. Understanding the immunogenicity of each component of a tetravalent vaccine is an important aspect of vaccine development and identifying appropriate correlates of protection, particularly because it is presently unclear how many serotype-specific responses will be required for protection from all four serotypes of DENV. However, dissecting the specific contribution of each element of a tetravalent vaccine is technically challenging due to the presence of antibodies that bind cross-reactive determinants shared by different components of the vaccine. A detailed map of the functionally important type-specific and group-reactive epitopes on the E protein is not presently available. Improved methodology that distinguishes and quantifies the functional contribution of each component of a tetravalent vaccine would allow for improvements in estimates of vaccine immunogenicity, for a more precise correlate of protection, and for a powerful investigational tool to study vaccine success and failure. To that end, we propose to develop a novel approach to measure the functional contributions of serotype-specific antibodies against the backdrop of a highly cross-reactive tetravalent polyclonal response. A panel of reporter virus particles (RVPs) containing mutations that destroy antibody-binding determinants will be used to identify functionally important epitopes that elicit type-specific or cross-reactive antibodies present in the serum of DENV vaccinees. An iterative screening process will guide selection of RVPs that are either no longer sensitive to neutralization by cross-reactive antibodies or type-specific responses. RVPs with these characteristics provide two complementary and specific methods for detecting type-specific responses in polyclonal sera as described below. These tools will then be employed to characterize the epitopes that comprise the neutralizing antibody response in vaccinated or naturally infected humans.