Anti-ICAM-i scFv from lactobacilli as a Microbicide

来自乳酸菌的抗 ICAM-i scFv 作为杀菌剂

• 批准号: 6809151
• 负责人: Richard B. Markham
• 金额: $ 47.21万
• 依托单位: OSEL, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6809151
• 关键词: HIV infections; Lactobacillus; SCID mouse; antiAIDS agent; biotechnology; biotherapeutic agent; cell adhesion molecules; communicable disease control; communicable disease transmission; cooperative study; crosslink; drug design /synthesis /production; drug vehicle; epithelium; human immunodeficiency virus 1; human tissue; immunoglobulin structure; plasmids; topical drug application; transfection /expression vector

Although the relative roles of cell-free and cell-associated HIV-1 in human sexual transmission are unknown, we have found using in vivo SCID mouse and in vitro transwell models of HIV-1 sexual transmission that cell-associated transmission of HIV-1 is highly efficient. These results differ from those reported in the macaque model system, which is likely accounted for by the fact that all of the reported macaque studies make no effort to adjust the environment to the neutral pH at which transmission occurs in the human setting. In our model systems, antibody to the cell adhesion molecule ICAM-1 is remarkably effective in blocking cell-associated transmission. This component of the Program Project proposal will therefore focus on using lactobacillus-based technology to block transmission of cell-associated virus. Preliminary data suggest that the antibody is acting by a mechanism that involves signal transduction rather than by simply blocking the docking function of ICAM-I. We are now hypothesizing that lactobacilli expressing single chain antibody fragments (scFv) can be used as a delivery mechanism for providing a sustained, totally transparent, and, in the case of heterosexual transmission, woman-controlled method for blocking cell-associated virus transmission. To evaluate this hypothesis we have established collaborations with the leading investigators in the world studying the expression of heterologous proteins by lactobacilli. In collaboration with these groups we seek 1) To examine using an in vitro transwell system the differential effects of intact Mab and Fab engagement of ICAM-1 in a cross-linking or a non-cross-linking manner on the movement of HIV-1 infected peripheral blood mononuclear cells across a human epithelial cell barrier. 2) To generate plasmids encoding human ICAM-l-specific scFv followed by their incorpporation into lactobacilli and subsequent testing of the efficacy of the secreted scFv or fixed lactobacilli expressing membrane-bound scFv in the in vitro transwell system. 3) To evaluate the protection efficacy of lactobacilli expressing anti-murine, anti-macaque, or anti-human ICAM-1 scFv in in vivo murine and macaque model systems of transmission of cell-associated and cell-free HIV-1. 4) To develop a chromosomally integrated expression system in Lactobacillus for expression of the selected scFv and subsequent testing in the murine and macaque models.

尽管无细胞和细胞相关 HIV-1 在人类性传播中的相对作用尚不清楚，但我们发现使用体内 SCID 小鼠和体外 HIV-1 性传播 Transwell 模型发现 HIV-1 的细胞相关传播效率很高。这些结果与猕猴模型系统中报告的结果不同，这可能是由于所有报告的猕猴研究都没有努力将环境调整到在人类环境中发生传播的中性 pH 值。在我们的模型系统中，细胞粘附分子 ICAM-1 的抗体在阻断细胞相关传播方面非常有效。因此，该计划项目提案的这一部分将重点关注使用基于乳酸菌的技术来阻止细胞相关病毒的传播。初步数据 表明该抗体通过涉及信号转导的机制发挥作用，而不是简单地阻断 ICAM-I 的对接功能。我们现在假设表达单链抗体片段（scFv）的乳杆菌可用作传递机制，以提供持续的、完全透明的、并且在异性传播的情况下由女性控制的方法来阻断细胞相关病毒的传播。为了评估这一假设，我们与世界上研究乳酸菌异源蛋白表达的领先研究人员建立了合作。与这些小组合作，我们寻求 1) 使用体外 Transwell 系统检查差异 以交联或非交联方式与 ICAM-1 结合的完整 Mab 和 Fab 对 HIV-1 感染的外周血单核细胞穿过人上皮细胞屏障的运动的影响。 2)产生编码人ICAM-1特异性scFv的质粒，随后将其掺入乳酸杆菌中，并随后在体外transwell系统中测试分泌的scFv或固定的表达膜结合scFv的乳酸杆菌的功效。 3) 评估表达抗鼠、抗猕猴或抗人ICAM-1 scFv的乳杆菌在细胞相关和无细胞HIV-1传播的体内鼠和猕猴模型系统中的保护功效。 4) 开发乳酸菌中的染色体整合表达系统，用于表达选定的 scFv 并在小鼠和猕猴模型中进行后续测试。