Trapping HIV in mucus with IgG antibodies

描述(申请人提供)：对于阴道传播，艾滋病毒必须通过粘液分泌物到达目标细胞；我们最近发现，艾滋病毒很容易通过pH中和的人宫颈阴道粘液传播。免疫系统产生的大多数抗体都分泌到粘液中(而不是血液或淋巴中)，当局部应用免疫球蛋白时，它可以在粘膜表面提供强大的抗艾滋病毒保护作用。然而，分泌或局部应用的免疫球蛋白如何预防粘液中的感染仍然知之甚少。我们假设，除了众所周知的抗体功能(如中和、补体激活、调理)外，免疫球蛋白还有一个未知的效应功能，即在粘液中捕获病毒。假设：通过与粘液凝胶形成弱的、短暂的亲和力，免疫球蛋白仍然可以自由地通过粘液扩散，并快速与病毒结合。当免疫球蛋白在病毒表面积聚时，免疫球蛋白的阵列可能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，可能处于亚中和免疫球蛋白浓度，可能会永久地将病毒困在粘液凝胶中。诱捕减少了到达靶细胞的病毒流量，并通过额外的保护机制促进了灭活和清除。粘液中这种潜在的免疫球蛋白捕获功能在很大程度上还没有被认识到，因为大多数关于粘液中的免疫球蛋白活性的研究还没有进行。在这项提议中，我们试图验证抗HIV免疫球蛋白会将HIV捕获到粘液中的假设。我们的初步观察表明，极低浓度的特定IgG1可以捕获单纯疱疹病毒和病毒样颗粒，否则它们会迅速穿透粘液凝胶。将艾滋病毒困在粘液中可能会减少或阻止艾滋病毒与靶细胞之间的任何有效接触。捕获的艾滋病毒将在性交后排出，和/或通过热降解、防御素和其他保护机制失活。如果确认为HIV，免疫球蛋白诱导的粘液捕获可能有助于解释亚中和浓度的粘膜保护，也可能有助于解释最近在泰国RV144艾滋病毒疫苗试验中观察到的适度保护。目的1：利用高分辨率粒子示踪技术，观察抗HIV免疫球蛋白中和人CVM中数百个HIV病毒粒子(带有GFP标签)的扩散速率，包括广谱中和B12、2G12、2F5和4E10以及非中和A32和50-69。量化艾滋病毒扩散(即诱捕效率)作为免疫球蛋白浓度和每个艾滋病毒病毒粒子结合的免疫球蛋白分子数量的函数。目的：测定抗HIV-IgS处理的人CVM与精浆中HIV的扩散速率，以及抗HIV-IgS单独处理的精浆中HIV的扩散速率。目的：确定粘液中的抗HIV-Ig G是否能阻止HIV病毒粒子到达并结合到硫酸肝素涂层表面，以模拟HIV与靶细胞上Syndecans的硫酸乙酰肝素的初始黏附步骤。这个项目将阐明粘液分泌物中的免疫球蛋白免疫保护的一个潜在的重要机制。这一结果可能有助于开发针对艾滋病毒的新型主动(疫苗)和被动(局部)免疫策略，利用免疫球蛋白将艾滋病毒捕获到粘液中的能力。 与公共卫生相关：艾滋病毒很容易通过pH中和的粘液分泌物传播感染。最近的证据表明，抗HIV抗体可以保护动物和人类免受粘膜HIV的挑战，但其保护机制仍然知之甚少。该项目将帮助设计主动(疫苗)和被动(局部)免疫战略，以帮助预防艾滋病毒感染。

DESCRIPTION (provided by applicant): For vaginal transmission, HIV must penetrate mucus secretions to reach target cells; we recently found that HIV readily diffuses through pH-neutralized human cervicovaginal mucus. Most antibodies produced by the immune system are secreted into mucus (not blood or lymph), and when IgG is applied topically it provides robust protection against HIV at mucosal surfaces. However, how secreted or topically applied IgG protects against infections in mucus remains poorly understood. We hypothesize that in addition to well-known antibody functions (e.g., neutralization, complement activation, opsonization), an unrecognized effector function of IgG is to trap viruses in mucus. Hypothesis: By forming weak, short-lived affinity bonds with the mucus gel, IgG is still free to diffuse through mucus and quickly bind to viruses. As IgG accumulates on the virus surface, the array of IgGs may form multiple low-affinity adhesive crosslinks between the virus and the mucus gel. A sufficient number of these low-affinity crosslinks, possibly at sub-neutralizing IgG concentrations, may permanently trap the virus in the mucus gel. Trapping reduces the flux of virus that reaches target cells, and facilitates inactivation and clearance by additional protective mechanisms. This potential IgG trapping function in mucus has been largely unrecognized because most studies of IgG activity have not been performed in mucus. In this proposal, we seek to test the hypothesis that anti-HIV IgGs will trap HIV in mucus. Our pilot observations indicate that remarkably low concentrations of specific IgG1can trap Herpes Simplex Virus and virus-like particles that otherwise rapidly penetrate mucus gels. Trapping HIV in mucus will likely reduce or block any effective contact between HIV and target cells. Trapped HIV will be shed with postcoital discharge, and/or become inactivated by thermal degradation, defensins, and other protective mechanisms. If confirmed for HIV, IgG-induced trapping in mucus may help explain mucosal protection at sub-neutralizing concentrations, and perhaps also help explain the modest protection observed in the recent RV144 Thai HIV- vaccine trial. Aim 1: Using high resolution particle tracking, observe diffusion rates of hundreds of HIV virions (with internal GFP tag) in neutralized human CVM treated with anti-HIV IgGs, including broadly neutralizing b12, 2G12, 2F5 and 4E10 as well as non-neutralizing A32 and 50-69. Quantify HIV diffusion (i.e. trapping efficiency) as a function of IgG concentration and number of IgG molecules bound per HIV virion. Aim 2: Measure diffusion rates of HIV in seminal plasma mixed with human CVM treated with anti-HIV IgGs, as well as in seminal plasma alone treated with anti-HIV-IgGs. Aim 3: Determine if anti-HIV IgG in mucus can block HIV virions from reaching and binding to heparan-sulfate coated surface to mimic the initial adhesive steps of HIV to heparan sulfates of syndecans on target cells. This project will clarify a potentially important mechanism of immune protection by IgG in mucus secretions. The results will likely aid in developing novel active (vaccine) and passive (topical) immunization strategies against HIV that exploit the ability of IgG to trap HIV in mucus. PUBLIC HEALTH RELEVANCE: HIV readily penetrates pH neutralized mucus secretions to transmit infection. Recent evidence suggests anti- HIV antibodies protect against mucosal HIV challenges in animals and humans, yet the mechanisms of protection remain poorly understood. This project will aid in engineering active (vaccine) and passive (topical) immunization strategies that may help protect against HIV infections.