Anti-ICAM-i scFv from lactobacilli as a Microbicide

虽然无细胞和细胞相关的HIV-1在人类性传播中的相对作用是未知的，我们已经发现使用体内SCID小鼠和体外transwell HIV-1性传播模型，HIV-1的细胞相关传播是非常有效的。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中性pH值，在该pH值下，人类环境中发生传播。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输中非常有效。因此，计划项目提案的这一部分将侧重于使用基于乳酸杆菌的技术来阻断细胞相关病毒的传播。初步数据 表明抗体是通过涉及信号转导的机制起作用的，而不是通过简单地阻断ICAM-1的对接功能。我们现在假设，表达单链抗体片段（scFv）的乳酸杆菌可用作提供持续的、完全透明的递送机制，并且在异性传播的情况下，可用作女性控制的方法来阻断细胞相关病毒传播。为了评估这一假设，我们与世界上研究乳酸杆菌表达异源蛋白的主要研究人员建立了合作关系。在与这些小组的合作中，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，然后将其掺入乳杆菌中，随后在体外transwell系统中测试分泌的scFv或表达膜结合scFv的固定乳杆菌的功效。3)评价表达抗鼠、抗猕猴或抗人ICAM-1 scFv的乳酸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)在乳杆菌中开发染色体整合的表达系统，用于表达所选的scFv并随后在小鼠和猕猴模型中进行测试。

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.