Methods to determine HIV Transport and Inactivation by Microbicide coating Layers

描述（由申请人提供）：该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病毒体从精液到组织的运输，并使杀微生物剂分子能够使病毒失效。我们将为杀微生物剂管道提供两种缺失的能力：（1）对涂层特性和抗病毒药物在中和精液传播的HIV中的相互作用的机理理解;以及（2）在动物和人类研究之前，帮助设计和比较产品的实用实验和数学工具。新的方法结合了两个相辅相成的程序。首先（具体目标1），我们将开发一种新的生物测定法，将一层载有HIV的精液（或精液模拟物）置于生物相关的薄杀微生物剂凝胶层上，该凝胶层置于组织外植体（或替代物）基质上。这种配置模拟阴道中的体内条件，我们假设，在杀微生物剂功效中起关键作用;在其凝胶载体内的杀微生物剂活性物的少数当前生物测定中未实现该配置。将测定HIV（总病毒和感染性病毒）向基质的转运-作为杀微生物剂产品层的功效的直接量度。该测定法还能够以比当前方法更现实的配置测量抗病毒剂从凝胶的释放动力学。第二（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 μ m）的长度尺度上，HIV和活性成分的杀微生物剂凝胶内的扩散系数。这些系数在控制病毒体从精液迁移到组织所需的时间方面至关重要。它们提供了关于凝胶涂层的结构特征如何影响病毒体和活性成分迁移率的信息。将测量的系数输入到HIV从精液到上皮的运输的机械数学模型，以及其被来自涂层的共扩散抗病毒剂（例如病毒包膜进入抑制剂）中和的机械数学模型。该模型提供了一种客观的方法来区分活性成分（浓度、效力、扩散系数）和涂层（厚度、HIV扩散系数）以及精液中的病毒载量在杀微生物剂产品效力中的相对作用。该模型可以帮助解释产品之间的差异 预测活性成分和递送载体的参数如何影响功效，并导致设计更好的产品。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重要的假设的新见解。他们将为杀微生物剂管道提供独特的药代动力学和药效学分析工具。 公共卫生相关性：该项目将创造新的，前所未有的方法来了解和测试杀微生物剂凝胶产品如何防止精液传播的艾滋病毒开始传播。这一方法将填补杀微生物剂管道中的一个空白，该管道正在寻求向妇女提供可以预防艾滋病毒和其他性传播病原体感染的产品。

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.