Development of transformed lactobacilli as a microbicide

描述（由申请人提供）：该提议的主要假设是，能够在广泛的女性横截面中持续阴道定殖的细菌可以被工程化以产生羊驼衍生的β整联蛋白特异性可变区抗体（VHH），其将抑制HIV-1感染。这种预防HIV-1传播的方法提供了靶向受体-配体相互作用的独特优势，这些相互作用完全独立于可变病毒蛋白。一种类似的针对单纯疱疹2型糖蛋白D的递送系统也将预防该病毒的感染，这增强了HIV-1感染的传播。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证明细菌递送的VHH的功效来追求该假设，其中一种是戈登链球菌，其被特别选择用于证明需要用孕酮预处理小鼠的小鼠模型系统中的原理证明的目的。鼠李糖乳杆菌GR-1将用于不需要孕酮预处理的小鼠模型中，因为这种菌株已被证明在广泛的女性群体中有效。乳酸杆菌作为杀微生物剂载体是有吸引力的，因为它们被FDA“通常认为是安全的”生物体，构成女性泌尿生殖道正常植物群的主要细菌组分，本质上抑制毒性更强的细菌的生长，对使用者是透明的，并且允许将杀微生物剂应用与性交分离。羊驼衍生的抗体结合结构域（VHH）是从一类骆驼抗体开发的，所述骆驼抗体仅由重链组成，并且其结合决定簇是单个蛋白质结构域，所述单个蛋白质结构域比可能由经典抗体产生的任何结构域小得多。利用这项技术，我们将：1）在羊驼中产生抗CD 18胞外域的抗体，CD 11 a/CD 18异源二聚体的β链组分是LFA-1。从免疫的羊驼的循环PBMC中，我们将产生通过噬菌体展示筛选结合亲和力的VHH文库。然后将从鼠李糖乳杆菌GR-1和戈登链球菌的质粒和/或细菌染色体表达那些VHH。2)使用目的1中描述的方法产生靶向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，评价抗CD 18和抗GpD的潜在毒性。4)在transwell或体外中和试验中检测CD 18和GpD特异性VHH阻断传播或中和感染的能力。5)评价转化的S. gordonii感染，并通过L.在非孕酮处理的人源化骨髓肝胸腺NOD-SCID（BLT）小鼠中的鼠李糖。评价定殖小鼠抵抗细胞相关和无细胞HIV-1感染的能力。6)评价转化的S.戈登孕酮处理的BALB/c小鼠。在这些小鼠中评价阴道灌洗液和血清中抗糖蛋白D和抗CD 18 VHH抗体的出现，并检查阴道灌洗液中VHH诱导的炎性细胞的出现。还将在该小鼠阴道攻击模型中测定原位产生的HSV-2特异性VHH的被动保护能力。和7）如果根据先前研究的结果，开发不依赖于抗生素选择的基于质粒的表达系统。在完成这些研究时，我们将使用体内模型系统证明该方法的潜在功效，并将开发可用于临床环境的VHH构建体，以及建立其从也可适用于临床环境的微生物物种的生产。

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.