Diffusion of viruses across human airway mucus and trapping by antibodies

说明（由申请人提供）：要感染肺部气道，病毒必须穿透粘液。然而，关于呼吸道病毒如何或如何通过呼吸道粘液（AM）扩散的效率，人们知之甚少。在本研究中，利用健康志愿者收集的人体AM，我们首先试图表征四种常见呼吸道病毒在新鲜AM体外的流动性，并确定哪些病毒容易穿透AM，哪些病毒被阻止或捕获。我们开发了不同尺寸的黏液惰性合成纳米探针，揭示了新鲜人黏液分泌物的网状间距（孔径）和纳米级粘弹性。因此，对于在AM中被减慢的病毒，我们可以确定有限的流动性是由位阻闭塞和/或黏液成分粘附引起的。最近，我们发现人类宫颈阴道粘液的网孔间距比哺乳动物病毒大得多，这与我们早期的观察一致，即HIV、HPV和诺瓦克病毒都很容易通过相同的粘液分泌物扩散。假设1:AM的网间距比大多数呼吸道病毒都大，如果病毒没有被粘合剂相互作用减缓，它们很容易穿透AM。因此，阻断肺部感染的一种方法是将病毒粘附在粘液中。大量研究表明，局部应用于粘膜表面（包括肺气道）的抗体（Ab）可以提供强大的抗感染保护，有些甚至在亚中和浓度下。免疫系统向粘液分泌的Ab比血液或淋巴分泌的Ab多，但粘液中Ab抵御感染的机制仍未得到充分研究。假设2：病毒结合的Ab阵列可以在病毒和黏液凝胶之间形成多个低亲和交联。足够数量的这种低亲和力交联，可能在亚中和浓度下，永久地将病毒困在粘液凝胶中。捕获减少了能够到达靶细胞的病毒粒子的通量，通过粘膜纤毛清除实现快速清除，并通过其他保护机制促进病毒降解和失活。我们的初步观察表明，非常低浓度的特异性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：测量常见呼吸道病毒（包括腺病毒、流感病毒和鼻病毒）在健康受试者体外AM中的传播率。我们还将表征AM的微观结构，以确定捕获的病毒是否通过位阻或粘附相互作用固定。目的2：确定分泌到天然AM中的病毒特异性抗体（IgG, IgA, sIgA, IgM）是否有助于AM中病毒的粘附捕获，并研究添加外源性病毒特异性IgG是否会捕获病毒，否则会迅速穿透AM。总之，Aim 1和Aim 2将提供呼吸道病毒如何穿透AM的定量描述，以及Ab如何通过在AM中捕获病毒来保护呼吸道。这一结果可能有助于开发新的疫苗或工程Ab，通过在AM中捕获病毒粒子来阻止肺部呼吸道的感染。

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.