Trapping HIV in mucus with IgG antibodies

描述（由申请人提供）：对于阴道传播，HIV必须穿透粘液分泌物到达靶细胞;我们最近发现HIV容易通过pH中和的人宫颈阴道粘液扩散。免疫系统产生的大多数抗体分泌到粘液中（不是血液或淋巴），当IgG局部应用时，它在粘膜表面提供了对HIV的强大保护。然而，分泌或局部应用的IgG如何保护粘液免受感染仍然知之甚少。我们假设，除了众所周知的抗体功能（例如，中和、补体激活、调理素作用），IgG的未被认识到的效应子功能是将病毒捕获在粘液中。假设：通过与粘液凝胶形成弱的、短暂的亲和键，IgG仍然可以自由地通过粘液扩散并迅速与病毒结合。随着IgG在病毒表面上积累，IgG阵列可以在病毒和粘液凝胶之间形成多个低亲和力粘附交联。足够数量的这些低亲和力交联，可能在亚中和IgG浓度下，可能将病毒永久捕获在粘液凝胶中。捕获减少了到达靶细胞的病毒通量，并通过额外的保护机制促进灭活和清除。粘液中这种潜在的IgG捕获功能在很大程度上尚未被认识到，因为大多数IgG活性的研究尚未在粘液中进行。在这项提议中，我们试图测试抗HIV IgG将HIV捕获在粘液中的假设。我们的初步观察表明，非常低浓度的特异性IgG 1可以捕获单纯疱疹病毒和病毒样颗粒，否则会迅速穿透粘液凝胶。将HIV捕获在粘液中可能会减少或阻止HIV与靶细胞之间的任何有效接触。被捕获的HIV将在性交后分泌物中脱落，和/或通过热降解、防御素和其他保护机制而失活。如果确认为HIV，IgG诱导的粘液捕获可能有助于解释亚中和浓度下的粘膜保护，也可能有助于解释在最近的RV 144泰国HIV疫苗试验中观察到的适度保护。目标1：使用高分辨率粒子追踪，观察用抗HIV IgG处理的中和人CVM中数百种HIV病毒粒子（具有内部GFP标签）的扩散速率，包括广泛中和的b12、2G 12、2F 5和4 E10以及非中和的A32和50-69。将HIV扩散（即捕获效率）定量为IgG浓度和每个HIV病毒体结合的IgG分子数量的函数。目标二：测量与用抗HIV IgG处理的人CVM混合的精浆中以及单独用抗HIV IgG处理的精浆中的HIV扩散率。目标3：确定粘液中的抗HIV IgG是否可以阻止HIV病毒粒子到达并结合硫酸乙酰肝素包被的表面，以模拟HIV与靶细胞上多配体聚糖的硫酸乙酰肝素的初始粘附步骤。该项目将阐明粘液分泌物中IgG的免疫保护的潜在重要机制。这些结果可能有助于开发新的主动（疫苗）和被动（局部）免疫策略，以利用IgG将HIV捕获在粘液中的能力。 公共卫生相关性：艾滋病毒容易穿透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的攻击，但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）免疫策略，可能有助于预防艾滋病毒感染。

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.