Chen Liyan, Williams Brent R, Yang Chiou-Ying, Cevallos Ana Maria, Bhat Najma, Ward Honorine, Sharon Jacqueline
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Int J Parasitol. 2003 Mar;33(3):281-91. doi: 10.1016/s0020-7519(02)00282-5.
The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C. parvum-specific polyclonal antibody libraries, standardised, perpetual mixtures of polyclonal antibodies, for which the genes are available. A combinatorial Fab phage display library was generated from the antibody variable region gene repertoire of mice immunised with C. parvum surface and apical complex glycoproteins which are believed to be involved in mediating C. parvum attachment and invasion. The variable region genes used to construct this starting library were shown to be diverse by nucleotide sequencing. The library was subjected to one round of antigen selection on C. parvum glycoproteins or a C. parvum oocyst/sporozoite preparation. The two selected libraries showed specific reactivity to the glycoproteins as well as to the oocyst/sporozoite preparation, with 50-73% antigen-reactive members. Fingerprint analysis of individual clones from the two antigen-selected libraries showed high diversity, confirming the polyclonality of the selected libraries. Furthermore, immunoblot analysis on the oocyst/sporozoite and glycoprotein preparations with selected library phage showed reactivity to multiple bands, indicating diversity at the antigen level. These C. parvum-specific polyclonal Fab phage display libraries will be converted to libraries of polyclonal full-length antibodies by mass transfer of the selected heavy and light chain variable region gene pairs to a mammalian expression vector. Such polyclonal antibody libraries would be expected to mediate effector functions and provide optimal passive immunity against cryptosporidiosis.
原生动物寄生虫微小隐孢子虫被视为全球主要的公共卫生问题，对免疫功能低下的个体尤其如此。尽管目前尚无有效的治疗方法，但特异性免疫反应可预防或终止隐孢子虫病，并且已发现被动给予的抗体可减轻感染的严重程度。因此，作为一种针对隐孢子虫病的免疫治疗方法，我们着手开发微小隐孢子虫特异性多克隆抗体文库，即标准化、永久性的多克隆抗体混合物，其基因是已知的。利用微小隐孢子虫表面和顶复合糖蛋白免疫小鼠，从其抗体可变区基因库中构建了一个组合Fab噬菌体展示文库，这些糖蛋白被认为参与介导微小隐孢子虫的附着和入侵。通过核苷酸测序表明，用于构建这个起始文库的可变区基因具有多样性。该文库在微小隐孢子虫糖蛋白或微小隐孢子虫卵囊/子孢子制剂上进行了一轮抗原筛选。两个筛选后的文库对糖蛋白以及卵囊/子孢子制剂均表现出特异性反应，其中50 - 73%的成员具有抗原反应性。对两个抗原筛选文库中单个克隆的指纹分析显示出高度多样性，证实了筛选文库的多克隆性。此外，用筛选文库噬菌体对卵囊/子孢子和糖蛋白制剂进行免疫印迹分析，结果显示对多条带具有反应性，表明在抗原水平上具有多样性。这些微小隐孢子虫特异性多克隆Fab噬菌体展示文库将通过将筛选出的重链和轻链可变区基因对大量转移到哺乳动物表达载体中，转化为多克隆全长抗体文库。预计这种多克隆抗体文库将介导效应功能，并提供针对隐孢子虫病的最佳被动免疫。
