Project 2: PREVENT Preclinical safety and efficacy studies

抗病毒凝集素GRFT(GRFT)具有广谱的抗人类免疫缺陷活性 病毒(HIV)1型和2型、单纯疱疹病毒2型(HSV-2)和丙型肝炎病毒(丙型肝炎病毒)(1-4)。 以格里菲辛为基础的局部杀微生物剂配方提供了作为一种多用途、非 以抗逆转录病毒为基础的预防艾滋病毒-1和流行病学相关感染的方法。《项目2》 目的：证明含有直肠杀微生物剂的GRFT治疗直肠结肠炎安全有效。 恒河猴HIV直肠传播模型。这些研究旨在 作为对项目3中提出的人体临床研究的补充。我们有三个具体目标： 目的1评价GRFT的安全性、药动学和药效学(PK/PD) 卡波姆凝胶。我们将在成年雄性恒河猴中进行三阶段安慰剂对照的PK/PD研究 使用GRFT直肠凝胶配方和由Core B提供的匹配安慰剂凝胶的猕猴。 拟议的猕猴研究旨在解决有关以下方面的具体翻译问题 GRFT暴露后粘膜环境的免疫毒性和健康。有利阶段1 结果包括检测直肠液中活性HIV-1抑制浓度GRFT和 可接受的临床安全指标，允许进入疗效阶段2。使用队列 GRFT治疗经验丰富的动物将使我们能够确定先前存在的免疫力是否对 GRFT影响产品功效。第三阶段将在感染SIV的患者中测试GRFT和安慰剂产品 我们将比较实验动物和对照动物体内的病毒排泄量， 提供了额外的安全指标。目标2是评估GRFT治疗对 粘膜免疫环境。我们将使用多重免疫分析和基因表达阵列 测定炎性免疫反应的标志物。高度敏感的 将应用在克里斯蒂娜·布罗利登教授的实验室中改进的免疫组织化学(IHC)技术 比较HIV靶细胞的数量、分布、激活状态和HIV的表达 受体靶向直肠上皮完整性的CD4、CCR5、DC-SIGN和Langerin+标记物 用GRFT活性药物或匹配的安慰剂治疗的动物的活检。流式细胞仪分析 还将对活检组织中的淋巴细胞进行计数和表型分析。目标3是评估 使用系统生物学方法对直肠粘膜环境进行GRFT治疗 利用现代质谱学蛋白质组学分析比较组织和直肠分泌物 来自接受活性药物和安慰剂的动物，并评估GRFT治疗的效果 对处理动物的直肠微生物区系的影响。

The antiviral lectin Griffithsin (GRFT) has broad spectrum activity against human immunodeficiency viruses (HIV) types 1 and 2, herpes simplex virus type 2 (HSV-2) and hepatitis C virus (HCV) (1-4). Griffithsin-based topical microbicide formulations offer the potential for use as a multipurpose, non- antiretroviral based method to prevent HIV-1 and epidemiologically linked infections. The Project 2 objective is to show that GRFT containing rectal microbicide products are safe and effective in the Rhesus macaque model of rectal HIV transmission. These studies are designed to be complementary to the human clinical studies proposed in Project 3. We have three specific aims: Aim 1 is to evaluate the safety, pharmacokinetics and pharmacodynamics (PK/PD) of GRFT carbopol gel. We will conduct a three stage placebo controlled PK/PD study in adult male Rhesus macaques with the GRFT rectal gel formulation and matched placebo gel supplied by Core B. The proposed macaque study is designed to address specific translational questions concerning immunotoxicity and health of the mucosal environment after GRFT exposure. Favorable stage 1 outcomes include detection of active HIV-1 inhibitory concentrations of GRFT in rectal fluids and acceptable clinical safety metrics, which allows progression to the efficacy Stage 2. Using a cohort of GRFT treatment experienced animals will allow us to determine whether pre-existing immunity to GRFT affects product efficacy. Stage 3 will test the GRFT and placebo products in SHIV infected animals, where we will compare amounts of virus shed in experimental versus control animals, providing an additional safety metric. Aim 2 is to assess the impact of GRFT treatment on the mucosal immune environment. We will use multiplexed immunoassays and gene expression arrays to measure markers of inflammatory immune responses. The highly sensitive immunohistochemistry (IHC) techniques refined in Prof. Kristina Broliden's laboratory will be applied to compare the number, distribution and activation state of HIV target cells and expression of HIV receptor targets CD4, CCR5, DC-SIGN and Langerin plus markers of epithelial integrity in rectal biopsies from animals treated with GRFT active drug or matched placebo. Flow cytometry analyses will also enumerate and phenotype lymphocytes in biopsy tissues. Aim 3 is to assess the impact of GRFT treatment on the mucosal environment in the rectum using a systems biology approach that utilizes modern mass spectrometry proteomics assays to compare tissues and rectal secretions from animals that receive active drug versus placebo, and to evaluate the effect of GRFT treatment on the rectal microbiota of treated animals.