Development of transformed lactobacilli as a microbicide

描述（由申请人提供）：本提案的主要假设是，能够在阴道内广泛定植的细菌可以通过工程设计产生羊驼衍生的β整合素特异性可变区抗体（VHH），从而抑制HIV-1感染。这种预防HIV-1传播的方法具有独特的优势，可以靶向完全独立于可变病毒蛋白的受体-配体相互作用。针对2型单纯疱疹糖蛋白D的类似递送系统也可以预防该病毒的感染，从而增强HIV-1感染的传播。这一假设将通过使用三种不同的小鼠模型系统和两种不同的细菌菌株来证明细菌递送的VHH的功效来实现，其中一种细菌菌株是戈登链球菌，专门用于证明需要用黄体酮对小鼠进行预处理的小鼠模型系统的原理证明。鼠李糖乳杆菌GR-1将用于不需要孕酮预处理的小鼠模型，因为这种乳酸菌菌株已被证明在广泛的女性横截面上定植是有效的。乳酸菌作为杀菌剂载体很有吸引力，因为它们被FDA“普遍认为是安全的”生物，构成了女性生殖泌尿道正常菌群的主要细菌成分，本质上抑制了更强毒性细菌的生长，对使用者来说是透明的，并且可以将杀菌剂与性交分离开来。羊驼衍生抗体结合域（VHH）是由一类仅由重链组成的骆驼类抗体发展而来的，其结合决定因子是单个蛋白结构域，比传统抗体产生的任何蛋白结构域都要小得多。利用这项技术，我们将：1)在羊驼中培养针对CD18外畴的抗体，CD11a/CD18异源二聚体的β链成分LFA-1。从免疫羊驼的循环PBMC中，通过噬菌体展示筛选VHH的结合亲和力，建立VHH文库。这些VHH随后将从鼠李糖乳杆菌GR-1和戈登链球菌的质粒和/或细菌染色体上表达。2)利用Aim 1中描述的方法生成靶向HSV-2糖蛋白d的VHH。3)通过测量两种不同体外模型系统的经皮细胞耐药性，评估抗cd18和抗gpd的潜在毒性。4)通过transwell或体外中和试验测试CD18和gpd特异性VHH阻断传播或中和感染的能力。5)评价转化链球菌在黄体酮处理的Hu-PBL-SCID小鼠阴道的定殖和cd18特异性VHH的分泌，鼠李糖在非黄体酮处理的人源骨髓肝胸腺NOD- SCID （BLT）小鼠体内的定殖。评估定植小鼠抵抗细胞相关和无细胞HIV-1感染的能力。6)评价转化黄体酮处理BALB/c小鼠阴道的定植和糖蛋白d特异性VHH的产生。评估这些小鼠阴道灌洗液和血清中抗糖蛋白D和抗cd18 VHH抗体的外观，并检查阴道灌洗液中由VHH诱导的炎症细胞的外观。在小鼠阴道攻击模型中，也将检测原位产生的HSV-2特异性VHH的被动保护能力。7)如果根据先前的研究结果，开发一种不依赖于抗生素选择的基于质粒的表达系统。在这些研究完成后，我们将利用体内模型系统证明这种方法的潜在功效，并将开发可用于临床环境的VHH结构，并建立其从乳酸菌物种生产的方法，也可能适用于临床环境。

DESCRIPTION (provided by applicant): The major hypothesis of this proposal is that bacteria capable of sustained vaginal colonization of a broad cross-section of women can be engineered to produce alpaca- derived beta integrin-specific variable region antibodies (VHH) that will inhibit infection with HIV-1. This approach to preventing HIV-1 transmission offers the unique advantage of targeting receptor-ligand interactions that are completely independent of mutable viral proteins. A similar delivery system targeting Glycoprotein D of herpes simplex type 2 will also prevent infection with that virus, which enhances transmission of HIV-1 infection. This hypothesis will be pursued by demonstration of the efficacy of bacterially-delivered VHH using three different mouse model systems and two different bacterial strains, one of which, Streptococcus gordonii, is selected specifically for the purpose of demonstrating proof of principle in mouse model systems that require pre-treatment of mice with progesterone. Lactobacillus rhamnosus GR-1 will be used in a mouse model that does not require progesterone pre-treatment, as this strain of lactobacillus has been demonstrated to be effective in colonizing a broad cross-section of women. Lactobacilli are appealing as a microbicide vehicle because they are "generally regarded as safe" organisms by the FDA, constitute the primary bacterial component of the normal flora of the female genitourinary tract, intrinsically inhibit the growth of more virulent bacteria, would be transparent to users, and would allow dissociation of microbicide application from coitus. Alpaca-derived antibody binding domains (VHH) are developed from a class of camelid antibodies consisting solely of heavy chains and the binding determinants of which are single protein domains that are much smaller than any which might be generated from classical antibodies. Using this technology we will: 1) Raise antibodies in alpacas against the ectodomain of CD18, the beta-chain component of the CD11a/CD18 heterodimer that is LFA-1. From circulating PBMC of the immunized alpacas we will generate a library of VHH screened by phage display for binding affinity. Those VHH will then be expressed from plasmids and/or the bacterial chromosome of Lactobacillus rhamnosus GR-1 and Streptococcus gordonii. 2) Using the methods described in Aim 1 generate VHH targeting HSV-2 glycoprotein D. 3) Evaluate the potential toxicity of anti-CD18 and anti-GpD by measuring transepithelial resistance in two different in vitro model systems. 4) Test the ability of the CD18 and GpD-specific VHH to block transmission or neutralize infection in transwell or in vitro neutralization assays. 5) Evaluate colonization of the mouse vagina and secretion of CD18-specific VHH by transformed S. gordonii in progesterone-treated Hu-PBL-SCID mice and by L. rhamnosus in non-progesterone-treated humanized bone marrow liver thymus NOD- SCID (BLT) mice. Evaluate the ability of colonized mice to resist infection with cell- associated and cell-free HIV-1. 6) Evaluate colonization of the mouse vagina and production of Glycoprotein D-specific VHH by transformed S. gordonii progesterone- treated BALB/c mice. Evaluate in these mice the appearance of anti-Glycoprotein D and anti-CD18 VHH antibodies in vaginal lavage fluid and serum and examine vaginal lavage fluid for the appearance of inflammatory cells induced by the VHH. The passive protective ability of in situ-produced HSV-2 specific VHH will also be assayed in this mouse vaginal challenge model. and 7) If dictated by results of the previous studies, develop a plasmid-based expression system that does not depend on antibiotic selection. At the completion of these studies, we will have proved the potential efficacy of this approach using in vivo model systems and will have developed the VHH constructs that could be used in the clinical setting as well as established their production from a lactobacillus species that also may be applicable to the clinical setting.