Diffusion of viruses across human airway mucus and trapping by antibodies

描述(申请人提供)：要感染肺部呼吸道，病毒必须穿透粘液。然而，关于呼吸道病毒如何或以何种效率在呼吸道粘液(AM)中传播，人们知之甚少。在这项建议中，我们首先利用从健康志愿者收集的人AM，在体外研究四种常见呼吸道病毒在新鲜AM中的迁移性，并确定哪些病毒容易穿透AM，哪些病毒被阻碍或捕获。我们开发了不同大小的粘液惰性合成纳米探针，揭示了新鲜人类粘液分泌物的网孔间距(孔径大小)和纳米级粘弹性。因此，对于在AM中减慢的病毒，我们可以确定有限的流动性是由空间闭塞和/或粘液成分粘连引起的。最近，我们发现人类宫颈阴道粘液中的网孔间距比哺乳动物病毒大得多，这与我们早期观察到的HIV、HPV和Norwalk病毒都很容易通过相同的粘液分泌物传播一致。假设1：AM中的网孔间距比大多数呼吸道病毒大，如果病毒不被粘附性相互作用减慢，它们很容易穿透AM。因此，阻止肺部感染的一种方法是将病毒粘在粘液中。大量研究表明，局部应用于粘膜表面的抗体(Ab)，包括肺部呼吸道，可以提供强大的预防感染的保护，一些甚至在亚中和浓度。免疫系统向粘液中分泌的抗体比血液或淋巴中分泌的抗体更多，但粘液中的抗体预防感染的机制尚不清楚。假设2：病毒结合的抗体阵列可以在病毒和粘液凝胶之间形成多个低亲和力的粘附性交联物。足够数量的这种低亲和力的交联物，可能处于亚中和浓度，永久地将病毒困在粘液凝胶中。诱捕减少了可到达靶细胞的病毒粒子的流量，通过粘液纤毛清除实现了快速消除，并通过其他保护机制促进了病毒的降解和灭活。我们的初步观察表明，极低浓度的特定IgG1可以捕获HSV和病毒样颗粒，否则它们会迅速穿透粘液凝胶。目的1：测定常见呼吸道病毒，包括腺病毒、流感病毒和鼻病毒在健康人AM体内的转运速率。我们还将对AM的微观结构进行表征，以确定捕获的病毒是否通过空间或粘附性相互作用固定。目的2：确定病毒特异性抗体(Ig G、Ig A、SIg A、Ig M)分泌到天然AM中是否能促进病毒在AM中的黏附捕获，并研究外源病毒特异性Ig G是否能捕获原本快速穿透AM的病毒AIM 1和AIM 2将一起提供呼吸道病毒如何穿透AM以及抗体如何通过捕获AM中的病毒来保护呼吸道的定量描述这一结果可能有助于开发新的疫苗或设计抗体，通过在AM中捕获病毒粒子来阻断肺部呼吸道的感染 与公共卫生相关：病毒必须穿透保护性粘液涂层才能感染粘膜上皮，但常见的呼吸道病毒如何克服和穿透人类呼吸道粘液(AM)屏障尚不清楚。局部应用于粘膜表面的抗体(Ab)通常能提供强大的预防感染的保护作用，但其机制尚不清楚。该项目的结果将有助于确定AM作为屏障减少感染的程度，AM的屏障特性是否依赖于分泌的抗体，以及AM屏障是否可以通过诱导分泌(即疫苗)或局部应用抗体来增强。

DESCRIPTION (provided by applicant): To infect lung airways, viruses must penetrate mucus. However, little is known about how or the efficiency with which respiratory viruses can diffuse across airway mucus (AM). In this proposal, using human AM collected from healthy volunteers, we first seek to characterize the mobility of four common respiratory viruses in fresh AM ex vivo, and identify which virus readily penetrates AM and which virus is hindered or trapped. We have developed different sized muco-inert synthetic nanoprobes that reveal the mesh spacing (pore size) and nanoscale viscoelasticity of fresh human mucus secretions. Thus, for viruses that are slowed in AM, we can determine whether the limited mobility is caused by steric occlusion and/or by adhesion to mucus constituents. Recently, we discovered that the mesh spacing in human cervicovaginal mucus is much larger than mammalian viruses, consistent with our earlier observations that HIV, HPV and Norwalk virus all readily diffuse through the same mucus secretions. Hypothesis #1: the mesh spacings in AM are larger than most respiratory viruses, and viruses will readily penetrate AM if they are not slowed by adhesive interactions. Thus, one approach to block pulmonary infections is to adhesively trap viruses in mucus. Numerous studies demonstrate that antibodies (Ab) applied topically to mucosal surfaces, including the lung airways, can provide robust protection against infections, some even at sub-neutralizing concentrations. The immune system secretes more Ab into mucus than blood or lymph, but the mechanisms by which Ab in mucus protect against infections remain poorly studied. Hypothesis #2: Array of virus-bound Ab can form multiple lowaffinity adhesive crosslinks between the virus and the mucus gel. A sufficient number of these low-affinity crosslinks, possibly at sub-neutralizing concentrations, permanently trap viruses in the mucus gel. Trapping reduces flux of virions that can reach target cells, enables rapid elimination via mucociliary clearance, and facilitates viral degradation and inactivation by other protective mechanisms. Our pilot observations indicate that remarkably low concentrations of specific IgG1 can trap HSV and virus-like particles that otherwise rapidly penetrate mucus gels. Aim 1: Measure the transport rates of common respiratory viruses, including adenovirus, influenza, and rhinoviruses, in human AM ex vivo obtained from healthy subjects. We will also characterize the microstructure of AM to determine whether trapped viruses that are immobilized by steric or adhesive interactions. Aim 2: Determine whether virus- specific Ab (IgG, IgA, sIgA, IgM) secreted into native AM may facilitate adhesive trapping of viruses in AM, and investigate whether addition of exogenous virusspecific IgG trap viruses that otherwise rapidly penetrate AM. Together, Aim 1 and 2 will provide a quantitative description of how respiratory viruses may penetrate AM, and how Ab may protect the airways by trapping viruses in AM. The results will likely aid in developing new vaccines or engineering Ab that block infections in the lung airways by trapping virions in AM. PUBLIC HEALTH RELEVANCE: Viruses must penetrate protective mucus coatings to infect mucosal epithelia, but how common respiratory viruses may overcome and penetrate human airway mucus (AM) barrier is unknown. Antibodies (Ab) applied topically to mucosal surfaces often provide robust protection against infections, but the mechanisms by which they do so remain poorly understood. The results in this project will aid in determining the extent to which AM acts as a barrier that reduces infection, whether the barrier properties of AM rely on secreted Ab, and if the AM barrier can be enhanced by induced secretion of (i.e. vaccine) or topically-applied Ab.