Trapping HIV in mucus with IgG antibodies

说明（由申请人提供）：阴道传播，艾滋病毒必须穿透粘液分泌物到达目标细胞；我们最近发现HIV很容易通过ph中和的人类宫颈阴道粘液扩散。免疫系统产生的大多数抗体分泌到粘液中（而不是血液或淋巴），当局部应用IgG时，它在粘膜表面提供强大的抗HIV保护。然而，分泌或局部应用IgG如何防止粘液感染仍然知之甚少。我们假设，除了已知的抗体功能（例如，中和，补体活化，活化），IgG的一个未被识别的效应功能是在粘液中捕获病毒。假设：通过与黏液凝胶形成弱的、短暂的亲和键，IgG仍然可以自由地通过黏液扩散并迅速与病毒结合。随着IgG在病毒表面的积累，IgG阵列可能在病毒和黏液凝胶之间形成多个低亲和力的黏附交联。足够数量的这些低亲和力交联，可能在亚中和的IgG浓度下，可能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通量，并通过额外的保护机制促进了失活和清除。这种潜在的IgG捕获功能在很大程度上未被认识到，因为大多数关于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困在粘液中的假设。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。被困住的HIV会随着性交后的分泌物而脱落，和/或通过热降解、防御和其他保护机制而失去活性。如果证实是HIV， igg诱导的黏液捕获可能有助于解释在亚中和浓度下的粘膜保护，也可能有助于解释在最近的RV144泰国HIV疫苗试验中观察到的适度保护。目的1：使用高分辨率粒子跟踪，观察数百种HIV病毒（带有内部GFP标签）在抗HIV igg处理的中和的人CVM中的扩散速率，包括广泛中和的b12, 2G12, 2F5和4E10以及非中和的A32和50-69。量化HIV扩散（即捕获效率）作为IgG浓度和每个HIV病毒粒子结合的IgG分子数的函数。目的2：测定HIV在经抗HIV igg治疗的人CVM混合精浆中以及单独经抗HIV- igg治疗的精浆中的扩散率。目的3：确定黏液中的抗HIV IgG是否可以阻止HIV病毒粒子到达并结合到硫酸肝素包被表面，以模拟HIV病毒在靶细胞上与syndecans的硫酸肝素的初始粘附步骤。本项目将阐明黏液分泌物中IgG免疫保护的潜在重要机制。这些结果可能有助于开发新的针对HIV的主动（疫苗）和被动（局部）免疫策略，利用IgG在粘液中捕获HIV的能力。

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.