Mechanisms of Antibody-Mediated Neutralization of Flavirus Infection

黄病毒是一组正链RNA病毒，由于其在人类和经济上重要的家养动物中的广泛分布和致病能力而具有全球性影响。该属的几个成员，如登革热病毒(DENV)和西尼罗河病毒(WNV)，被认为是新出现或重新出现的病原体，因为它们与人类接触并导致疾病的发病率每年以惊人的速度增加。每年有5000多万人感染黄病毒。 西尼罗河病毒是该属的蚊媒成员，是西尼罗河脑炎的病原体。西尼罗河病毒在非洲、澳大利亚、欧洲、亚洲和中东的部分地区流行，是人和马脑炎周期性暴发的原因。1999年西尼罗河病毒传入北美，并在美国迅速传播到加拿大、墨西哥和加勒比海地区，确认该病毒是一种对西半球具有临床和经济意义的新病原体。虽然血清阳性率研究表明，人类的大多数西尼罗河病毒感染是亚临床的，但临床上的明显感染范围从发热性疾病(西尼罗热)到更严重和可能致命的神经系统疾病。目前，还没有西尼罗河病毒疫苗被批准用于人类，治疗是支持性的。 冷冻电子显微镜研究表明，黄病毒病毒粒子表面被高度有序的二十面体覆盖，该二十面体由180种被膜蛋白组成。产生能够与这种病毒蛋白结合并阻断感染的抗体是免疫反应的关键方面，也是疫苗开发的重要目标。被动免疫研究和对免疫缺陷小鼠的实验感染表明，抗体在抵御黄病毒感染方面发挥着重要作用。体内抗体的重要性反映了直接中和病毒感染性的能力，以及由抗体分子的恒定部分介导的几种不同的效应功能。 抗体的中和潜力取决于病毒粒子上可结合的位置的数量(由表位可及性决定)和结合的强度(抗体亲和力/亲和力)。利用中和抗体结合西尼罗河病毒或登革热病毒病毒粒子上不同的结构部位，我们正在研究抗体如何与病毒颗粒结合以及抗体与病毒颗粒结合的数量的有效性的生化基础。我们最近的数据表明，中和西尼罗河病毒需要单个病毒粒子与超过所需阈值的化学计量比接触。我们对这一门槛的估计是大约30种抗体。 由于成熟病毒粒子的准二十面体对称性，并不是黄病毒上的所有表位都同样能与抗体结合。可及性调节抗体效力，因为一些决定因素可能不会以允许中和的频率暴露。因此，识别这些表位的抗体可能中和很差，甚至根本不中和，即使在允许饱和的浓度下也是如此，因为太少的抗体可以同时对接在病毒粒子上。有趣的是，许多识别成熟病毒粒子上暴露较差表位的抗体在体外仍然显示出中和活性，并在体内发挥保护作用。抗体如何使病毒粒子上的神秘表位与允许中和的化学计量学相结合，很难使用现有的病毒粒子结构和包膜组织的静态模型进行协调。为了研究控制针对隐蔽表位的抗体效力的机制，我们正在调查控制表位可及性和中和效力的动力学。 抗体依赖的感染增强。 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，随着孩子年龄的增长，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，并在体外增强Fc受体表达细胞的病毒感染。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。为了促进这一目标，我们的实验室正在研究构成抗体增强特性的生化决定因素，以及作为ADE机制基础的细胞生物学。

Flaviviruses are a group of positive-stranded RNA viruses that have a global impact due to their widespread distribution and ability to cause disease in humans and economically important domesticated animals. Several members of this genus, such as dengue virus (DENV) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because the incidence with which they encounter humans and cause disease is increasing each year at an alarming rate. There are more than 50 million humans infected by flaviviruses each year. WNV is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. WNV is endemic in parts of Africa, Australia, Europe, Asia, and the Middle East and has been responsible for periodic outbreaks of encephalitis in humans and horses. The introduction of WNV into North America in 1999 and its rapid spread across the United States into Canada, Mexico, and the Caribbean identifies this virus as an emerging pathogen of clinical and economic significance for the Western Hemisphere. While seroprevalence studies indicate that most WNV infections of humans are subclinical, clinically apparent infections range from a febrile illness (West Nile fever) to more severe and potentially fatal neurologic disease. Currently, no WNV vaccine has been approved for use in humans and treatment is supportive. Cryo-electron microscopy studies reveal that the surface of flavivirus virions is covered by a highly ordered icosahedron composed of 180 envelope proteins. The generation of antibodies capable of binding to this array of viral proteins and blocking infection is a critical aspect of the immune response and an important goal of vaccine development. Passive immunization studies and experimental infections of immunodeficient mice demonstrate that antibody plays a significant role in protection from flavivirus infection. The importance of antibodies in vivo reflects an ability to directly neutralize virus infectivity, as well as several distinct effector functions mediated by constant portions of the antibody molecule. The neutralization potential of an antibody is governed by the number of sites on the virion available for binding (determined by epitope accessibility) and the strength of binding (antibody affinity/avidity). Using neutralizing antibodies that bind structurally distinct sites on the WNV or DENV virion, we are investigating the biochemical basis of potency with respect to how antibodies engage virus particles, and in what numbers. Our recent data suggest that neutralization of WNV requires engagement of individual virions with a stoichiometry that exceeds a required threshold. Our estimate for this threshold is roughly 30 antibodies. Due to the quasi-icosahedral symmetry of the mature virion, not all epitopes on the flavivirus are equally accessible for antibody binding. Accessibility modulates antibody potency, as some determinants may not be exposed with a frequency that permits neutralization. Thus, antibodies that recognize such epitopes may neutralize poorly, or not at all, even at concentrations that permit saturation because too few antibodies can simultaneously dock on the virion. Of interest, many antibodies that recognize poorly exposed epitopes on the mature virion still show neutralizing activity in vitro and protect in vivo. How antibodies engage cryptic epitopes on virions with a stoichiometry that permits neutralization is difficult to reconcile using existing static models of virion structure and envelope organization. To investigate mechanisms that govern the potency of antibodies that target cryptic epitopes, we are investigating the dynamics that control epitope accessibility and neutralization potency. Antibody-dependent enhancement of infection. 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 of Fc-receptor-expressing cells 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. To facilitate this goal, our laboratory is investigating the biochemical determinants that comprise the enhancing character of an antibody, and the cell biology that underlies the mechanism of ADE.