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一种杀微生物剂产品阴道上皮涂层的功能--抑制艾滋病毒病毒粒子从精液到组织的传输，并使杀微生物剂分子能够禁用病毒。我们将为杀微生物剂管道贡献两项缺失的能力：(1)在中和精液中和艾滋病毒方面，对涂层性能和抗病毒制剂之间相互作用的机械理解；(2)在动物和人体研究之前，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层杀菌剂凝胶层上，该凝胶层位于组织外植体(或代用品)底物上。这种配置模拟了阴道内的活体条件，我们假设，这种条件对杀微生物剂的效果起着关键作用；目前在凝胶载体内进行的为数不多的杀微生物活性生物测试中，这种配置是无法实现的。将确定艾滋病毒(总病毒和传染性病毒)向底物的传输--作为杀微生物剂产品层效力的直接衡量标准。该方法还可以测量凝胶中抗病毒药物的释放动力学，比目前的方法更接近实际情况。第二(具体目标2)我们将开发一种技术来确定HIV和活性成分的杀微生物剂凝胶中的扩散系数，其覆盖范围是在人类阴道中测量的涂层厚度(100-500 5m)。这些系数对控制病毒粒子从精液迁移到组织所需的时间至关重要。它们提供了有关凝胶涂层的结构特征如何影响病毒粒子和活性成分迁移率的信息。测量的系数将被输入到艾滋病毒从精液到上皮的传输的机械数学模型中，以及通过从涂层共扩散抗病毒药物(例如病毒包膜进入抑制剂)而中和的机制数学模型。该模型为区分有效成分(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菌剂产品疗效中的相对作用提供了客观的手段。该模型可以帮助解释产品之间的差异，预测有效成分和输送工具的参数如何影响疗效，并导致设计出更好的产品。特定的目标1和2是协同的，为上皮涂层--最初的和持续的--对半固体杀菌剂产品功效重要的假设提供了新的见解。他们将为杀菌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、史无前例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艾滋病毒。这一方法将填补杀微生物剂管道中的一个空白，该管道正寻求为女性提供能够预防感染艾滋病毒和其他性传播病原体的产品。

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.