Mechanisms of Antibody-Mediated Neutralization of Flavirus Infection

黄病毒是一组正链RNA病毒，由于其广泛分布和在人类和经济上重要的驯养动物中引起疾病的能力而具有全球影响。该属的几个成员，如登革病毒（DENV）和西尼罗河病毒（WNV），被认为是新兴或重新出现的病原体，因为它们遇到人类并引起疾病的发病率每年以惊人的速度增加。每年有超过5000万人感染黄病毒。 西尼罗河病毒是蚊媒传播的成员，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，并导致人类和马脑炎的周期性爆发。西尼罗河病毒于1999年传入北美，并迅速在美国传播到加拿大、墨西哥和加勒比地区，这表明该病毒是西半球具有临床和经济意义的新兴病原体。虽然血清阳性率研究表明，大多数人感染西尼罗河病毒是亚临床的，临床上明显的感染范围从发热性疾病（西尼罗河热）到更严重和可能致命的神经系统疾病。目前，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，黄病毒的病毒粒子表面覆盖着一个高度有序的二十面体，由180个包膜蛋白组成。产生能够结合这种病毒蛋白并阻断感染的抗体是免疫应答的一个关键方面，也是疫苗开发的一个重要目标。被动免疫研究和免疫缺陷小鼠的实验感染表明，抗体在保护免受黄病毒感染中起重要作用。抗体在体内的重要性反映了它们直接中和病毒感染性、促进补体介导的病毒体裂解和通过Fc受体依赖性途径促进有效的病毒清除的能力。然而，响应于自然感染或疫苗接种而产生的抗体也具有在体外和体内增强病毒感染的潜力。 抗体的中和潜力取决于病毒体上可用于结合的位点的数量（由表位可及性确定）和结合强度（抗体亲和力/亲合力）。使用中和抗体，结合结构上不同的网站上的WNV或DENV病毒粒子，我们正在调查的生化基础上的效力，抗体如何参与病毒颗粒，并在什么数量。我们最近的数据表明，中和西尼罗河病毒需要与个别病毒粒子的化学计量，超过所需的阈值。我们估计这个阈值大约是30个抗体。 由于成熟病毒粒子的准二十面体对称性，并非黄病毒上的所有表位都同样可用于抗体结合。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，即使在允许饱和的浓度下，识别这些表位的抗体也可能中和不良，或根本不中和，因为太少的抗体可以同时停靠在病毒体上。巧合的是，许多识别成熟病毒体上暴露较差的表位的抗体在体外和体内仍显示出中和活性。抗体如何以允许中和的化学计量接合病毒体上的隐蔽表位，难以使用病毒体结构和包膜组织的现有静态模型来调和。为了研究控制靶向隐蔽表位的抗体效力的机制，我们正在研究控制表位可及性和中和效力的动力学。 抗体依赖性感染增强。 巧合的是，抗体也可能在增强病毒感染和加重疾病中发挥作用。抗体依赖性感染增强（ADE）描述了在亚中和浓度的抗体或免疫血清存在下携带Fc受体的细胞的感染的显著增加。ADE与DENV感染的临床结局之间最直接的联系来自于对出生后第一年内在婴儿中观察到的原发性感染后异常大量DHF病例的调查。出生时，DENV特异性被动获得抗体以相对高的浓度存在，并在体外表现出中和活性。然而，随着儿童年龄的增长，母体获得性抗体的降解继续达到不再具有保护作用的水平，不能中和病毒，并增强体外病毒感染。婴儿的抗体滴度下降到体外支持ADE的水平与生命第一年期间原发性DENV感染后DHF的风险平行。在更广泛的背景下，由一种血清型的DENV的初次感染引起的抗体可以结合在二次感染期间引入的相关病毒，其亲和力降低，导致病毒粒子以不允许病毒中和但可以支持ADE的化学计量接合。产生免疫应答，提高疫苗中存在的所有四种血清型病毒的中和抗体的保护水平，是降低ADE风险的关键因素。为了实现这一目标，我们的实验室正在研究组成抗体增强特性的生化决定因素，并研究ADE机制的细胞生物学基础。

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 their ability to directly neutralize virus infectivity, facilitate complement-mediated lysis of virions, and promote efficient viral clearance through Fc-receptor dependent pathways. However, antibodies generated in response to natural infection or vaccination also have the potential to enhance virus infection both in vitro and in vivo. 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. Paradoxically, many antibodies that recognize poorly exposed epitopes on the mature virion still show neutralizing activity in vitro and 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 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 studying the cell biology that underlies the mechanism of ADE.