Development of transformed lactobacilli as a microbicide

描述(申请人提供)：这项建议的主要假设是，能够在大范围的妇女中持续阴道定植的细菌可以被改造成产生羊驼β整合素特异性可变区抗体(VHH)，从而抑制HIV-1的感染。这种预防HIV-1传播的方法提供了针对受体-配体相互作用的独特优势，这些相互作用完全不依赖于可变的病毒蛋白。针对单纯疱疹病毒2型糖蛋白D的类似递送系统也将防止感染该病毒，从而增强HIV-1感染的传播。这一假说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传递的VHH的有效性，其中一种细菌，戈登链球菌，被特别选择的目的是为了在需要对小鼠进行孕酮预治疗的小鼠模型系统中证明原理的证明。鼠李糖乳杆菌gr-1将用于不需要孕激素预治疗的小鼠模型，因为这种乳杆菌已被证明在广泛的女性群体中定植有效。乳酸菌作为一种杀微生物剂很有吸引力，因为它们被美国食品和药物管理局“普遍认为是安全的”生物体，构成了女性生殖道正常菌群的主要细菌成分，本质上抑制了毒力更强的细菌的生长，对使用者是透明的，并允许将杀菌剂的应用与性行为分开。羊驼来源的抗体结合域(VHH)是由一类完全由重链组成的骆驼抗体发展而来的，其结合决定簇是单个蛋白结构域，比任何经典抗体产生的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胞外区的抗体，CD18是CD11a/CD18异源二聚体的β链成分，即LFA-1。通过噬菌体展示技术筛选免疫羊驼外周血中的外周血单核细胞，构建VHH文库，进行亲和力筛选。然后，这些VHH将从鼠李糖乳杆菌GR-1和戈登链球菌质粒和/或细菌染色体中表达。2)使用AIM 1中描述的方法制备针对HSV-2糖蛋白D的VHH。3)通过在两种不同的体外模型系统中测量跨上皮阻力来评估抗CD18和抗GPD的潜在毒性。4)在Transwell或体外中和试验中检测CD18和GPD特异性VHH阻断传播或中和感染的能力。5)评价转化的戈登葡萄球菌在孕酮处理的Hu-PBL-SCID小鼠和鼠李糖在非孕酮处理的人源化骨髓肝胸腺nod-SCID(BLT)小鼠体内的定植和CD18特异性VHH的分泌。评估定植的小鼠抵抗细胞相关和无细胞HIV-1感染的能力。6)观察转化的戈登螺旋体孕酮处理的BALB/c小鼠在阴道的定植和产生糖蛋白D特异性的VHH。评估小鼠阴道灌洗液和血清中抗糖蛋白D和抗CD18 VHH抗体的出现情况，并检查阴道灌洗液中VHH诱导的炎症细胞的出现情况。在这个小鼠阴道攻击模型中，还将检测原位产生的HSV-2特异性VHH的被动保护能力。7)如果之前的研究结果所规定的，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，我们将使用体内模型系统来证明这种方法的潜在有效性，并将开发出可用于临床环境的VHH结构，以及建立其从也可能适用于临床环境的乳酸菌物种中产生的VHH结构。

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.