Methods to determine HIV Transport and Inactivation by Microbicide coating Layers

描述（由申请人提供）：本项目将开发和应用方法来分析杀微生物剂产品阴道上皮涂层的功能-抑制HIV病毒粒子从精液到组织的运输，并使杀微生物剂分子使病毒失效。我们将为杀微生物剂管道提供两个缺失的能力：(1)对涂层特性和抗病毒药物在中和精液HIV中的相互作用的机制理解；(2)实用的实验和数学工具，以帮助设计和比较产品，在动物和人体研究之前。新方法结合了两个互补的程序。首先（具体目标1），我们将开发一种新的生物测定方法，将一层携带艾滋病毒的精液（或精液模拟物）放置在生物相关的薄杀菌剂凝胶层上，该凝胶层位于组织外植体（或替代物）底物上。这种结构模拟了阴道的体内条件，我们假设，在杀微生物剂的功效中起关键作用；目前在凝胶载体内对杀微生物剂活性进行的少数生物测定中，没有实现这种配置。将确定HIV（总病毒和传染性病毒）到底物的运输情况——作为杀微生物剂产品层有效性的直接衡量标准。该分析还可以测量凝胶中抗病毒药物的释放动力学，比目前的方法更现实。其次（具体目标2），我们将开发一种技术，以确定在人类阴道中测量的涂层厚度（100 - 500米）的长度范围内，HIV和有效成分的杀菌剂凝胶内的扩散系数。这些系数对于控制病毒粒子从精液迁移到组织所需的时间至关重要。它们提供了关于凝胶涂层结构特征如何影响病毒粒子和活性成分流动性的信息。测量的系数将输入到HIV从精液转运到上皮的机械数学模型中，以及通过包膜层的共扩散抗病毒药物（例如病毒包膜进入抑制剂）来中和HIV。该模型为区分有效成分（浓度、效价、扩散系数）和包衣层（厚度、HIV扩散系数）以及精液中病毒载量对杀菌剂产品功效的相对作用提供了一种客观手段。该模型有助于解释产品之间的差异，预测有效成分参数和递送载体对功效的影响，从而设计出更好的产品。具体目标1和2是协同的，提供了关于上皮涂层-初始和持续-对半固体杀微生物剂产品功效很重要的假设的新见解。它们将为杀微生物剂管线提供独特的药代动力学和药效学分析工具。

DESCRIPTION (provided by applicant): This project will develop and apply methods to analyze functioning of a microbicide product vaginal epithelial coating layer - to inhibit transport of HIV virions from semen to tissue and enable microbicide molecules to disable virus. We will contribute two missing capabilities to the microbicide pipeline: (1) mechanistic understanding of interactions between coating properties and anti-viral agents in neutralizing semen-born HIV; and (2) practical experimental and mathematical tools to help design and compare products, prior to animal and human studies. The new methodology combines two complementary procedures. First (Specific Aim 1) we will develop a new bioassay that places a layer of HIV-laden semen (or semen simulant) over a biologically relevant thin microbicide gel layer that rests on a tissue explant (or surrogate) substrate. This configuration simulates in vivo conditions in the vagina that, we hypothesize, play a critical role in microbicide efficacy; the configuration is not achieved in the few current bioassays of microbicide actives within their gel vehicles. Transport of HIV (total virus and infectious virus) to the substrate will be determined - as a direct measure of efficacy of the microbicide product layer. This assay also enables measurement of release kinetics of antiviral agents from the gels, in a more realistic configuration than current methods. Second (Specific Aim 2) we will develop a technique to determine diffusion coefficients within microbicide gels of HIV and active ingredients over the length scales of coating thickness (100 - 500 5m) that have been measured in the human vagina. These coefficients are paramount in governing the time required for virions to migrate from semen to tissue. They provide information about how structural characteristics of a gel coating can impact virion and active ingredient mobilities. Measured coefficients will be input to a mechanistic mathematical model of HIV transport from semen to epithelium, and of its neutralization by co- diffusing antiviral agents from a coating layer, e.g. viral envelope entry inhibitors. The model provides an objective means to distinguish the relative roles of the active ingredient (concentration, potency, diffusion coefficient) and coating layer (thickness, HIV diffusion coefficient), together with viral load in semen, in microbicide product efficacy. The model can help interpret differences between products predicting how parameters of active ingredients and delivery vehicles affect efficacy, and leading to design of better products. Specific Aims 1 and 2 are synergistic, providing new insights about the hypothesis that epithelial coating - initial and sustained - is important to semi-solid microbicide product efficacy. They will contribute unique pharmacokinetic and pharmacodynamic analysis tools to the microbicide pipeline. PUBLIC HEALTH RELEVANCE: This project will create novel, unprecedented methodology to understand and test how microbicide gel products prevent semen-borne HIV from initiating transmission. This methodology will fill a gap in the microbicide pipeline, which is seeking to provide women with products that can prevent infection by HIV and other sexually transmitted pathogens.