POLYCLONAL ANTIBODY LIBRARIES FOR C PARVUM THERAPY

该项目的目标是开发一种安全有效的治疗癌症的药物。 寄生虫机会性感染的治疗或预防 微小隐孢子虫(C.parvum)在艾滋病患者中，这种寄生虫会导致 严重的慢性腹泻，体液和体重减轻，经常死亡，以及 目前还没有已知的有效治疗方法。我们建议开发 多克隆抗体库将用于被动免疫治疗 隐孢子虫病(微小隐孢子虫感染)。多克隆抗体库 结合了靶向多个抗原决定簇的优势(低 抗原逃逸变异体的可能性与效应物的有效调节 功能)，具有使用单抗的优点(无限制 标准化试剂的供应和遗传基因的可用性 用于所需操作的材料)。 这项建议的具体目标是建立多克隆文库 人和鼠-人嵌合的抗C. 并检测多克隆抗体库在人类免疫缺陷病毒感染中的作用 在体外，S还在体内建立了环孢子虫病的动物模型。 为了产生多克隆抗体库，Fab噬菌体展示文库 将首先通过重组DNA技术从免疫的小鼠中创建 不同发育阶段的微小隐孢子虫，以及来自人类的高致病性 对微小弧菌的抗体水平。Fab噬菌体展示文库将是 对微小隐孢子虫发育阶段的反应性阳性选择和 负面选择以消除对人类和小鼠的交叉反应 组件。猪瘟病毒重链可变区和轻链可变区基因对 选定的Fab噬菌体展示文库将批量转移到 表达完整IgA多克隆文库的哺乳动物表达载体 以及全部为人类、纯小鼠或鼠-人嵌合体的抗体。 免疫球蛋白A多克隆文库也将转化为分泌型免疫球蛋白A(SIgA)。 文库与分泌成分的体外结合。所有的 图书馆将在生产的不同阶段进行效果评估 对微小弧菌的体外检测，包括ELISA、免疫荧光、 免疫印迹分析及其对微小弧菌附着和侵袭的抑制作用 上皮细胞。还将测试Ig G、Ig A和SIGA库 当口服和非肠道给药给药时的疗效- 严重联合免疫缺陷(SCID)小鼠模型的感染 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.