POLYCLONAL ANTIBODY LIBRARIES FOR C PARVUM THERAPY

该项目的目标是开发一种安全有效的药物， 治疗或预防寄生虫的机会性感染 隐孢子虫（Cryptosporidium parvum）。parvum）。 在艾滋病患者中，这种寄生虫会导致 严重的慢性腹泻伴液体和体重减轻，经常死亡， 目前还没有已知的有效治疗方法。 我们建议发展 多克隆抗体文库用作被动免疫疗法， 隐孢子虫病（C.细小病毒感染）。 多克隆抗体库 联合收割机结合靶向多个抗原决定簇（低 抗原逃逸变体的可能性和效应子的有效介导 功能）的优点，使用单克隆抗体（无限 标准化试剂的供应和遗传学 用于所需操作的材料）。 该建议的具体目标是产生以下的多克隆文库： 特异于C. 并测试多克隆抗体库在 体外和体内在猪孢子虫病的动物模型中。 为了产生多克隆抗体文库，Fab噬菌体展示文库 将首先通过重组DNA技术从免疫小鼠中产生 C.小，从人类与高 C.抗体水平小的 Fab噬菌体展示文库将在 对C.小孢子发育阶段， 阴性选择，以消除对人和小鼠的交叉反应性 件. 重组质粒的重链可变区和轻链可变区基因对分别与重组质粒的重链可变区和轻链可变区基因对进行同源重组。 选择的Fab噬菌体展示文库将被批量转移至 表达完整伊加多克隆文库的哺乳动物表达载体 和IgG抗体，它们都是人、全小鼠或小鼠-人嵌合体。 伊加多克隆文库也将被转化为分泌型伊加（sIgA）。 通过与分泌成分的体外结合来构建文库。 所有的 将在生产的各个阶段对文库的有效性进行评估 针对C.通过包括ELISA，免疫荧光， 免疫印迹分析和C.寄生侵入 的上皮细胞。 还将检测IgG、伊加和sIgA文库 当口服和胃肠外给药于C.微小的，微小的 严重联合免疫缺陷（SCID）小鼠模型中感染的小鼠， 隐孢子虫病

The goal of this project is to develop a safe and effective drug for the treatment or prevention of opportunistic infection with the parasite Cryptosporidium parvum (C. parvum). In AIDS patients, this parasite causes severe chronic diarrhea with fluid and weight loss, and often death, and there is currently no known effective treatment. We propose to develop polyclonal antibody libraries to be used as passive immunotherapy for Cryptosporidiosis (C. parvum infection). Polyclonal antibody libraries combine the advantages of targeting multiple antigenic determinants (low likelihood of antigen escape variants' and efficient mediation of effector functions) with the advantages of using monoclonal antibodies (unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations). The specific aims of this proposal are to generate polyclonal libraries of human and mouse-human chimeric IgA and IgG antibodies specific for C. parvum and to test the efficacy of the polyclonal antibody libraries in vitro, s well as in vivo in an animal model of cyrptosporidiosis. To generate the polyclonal antibody libraries, Fab phage display libraries will first be created by recombinant Dna techniques from mice immunized with various developmental stages of C. parvum, and from humans with high antibody levels to C. parvum. The Fab phage display libraries will be positively selected for reactivity to C. parvum developmental stages and negatively selected to remove cross-reactivities to human and mouse components. The heavy and light chain variable region gene pairs of the selected Fab phage display libraries will be transferred in bulk to mammalian expression vectors to express polyclonal libraries of intact IgA and IgG antibodies that are all human, all-mouse, or mouse-human chimeras. The IgA polyclonal libraries will also be converted to secretory IgA (sIgA) libraries by in vitro association with secretory component. All the libraries will be evaluated, at various stages of production, for efficacy against C. parvum by in vitro assays including ELISA, immunofluorescence, immunoblot analysis, and inhibition of C. parvum attachment to and invasion of epithelial cells. The IgG, IgA, and sIgA libraries will also be tested for efficacy when administered orally and parenterally to C. parvum- infected mice in a severe combined immunodeficiency (SCID) mouse model of cryptosporidiosis.