Characterization of therapeutic human monoclonal antibodies against SARS

治疗性人抗SARS单抗的鉴定 冠状病毒是SARS的病原体及其快速基因组测序的发现为开发有效的预防和治疗这种病毒的方式创造了机会。特别令人感兴趣的是尖峰糖蛋白(S蛋白)，它以表面投影的形式表达。S蛋白参与病毒附着、细胞间融合、病毒保护性免疫反应的诱导 中和。这项提议将侧重于开发可用于预防和/或暴露后治疗SARS冠状病毒感染的人类单抗。我们已经利用他们的XenoMouse与Abgenix Inc.密切合作，产生了一组能够产生中和人类单抗(HmAbbs)的杂交瘤细胞。这些杂交瘤已被克隆，19株HmAb已被生产、纯化并在中和试验中重新测试。此外，我们已经制备了S蛋白的几个重组片段，并获得了跨越S蛋白的重叠多肽，并用它们来确定HmAbs的结合特异性。在AIM-1中，我们将进一步鉴定人抗SARS-CoV单抗的体外中和能力，并使用一些不同的当前方法来确定它们的相对结合亲和力和特异性。在AIM-2中，我们将鉴定能够潜在中和广泛的 通过病毒进入和细胞融合试验对临床分离株进行检测，这些伪型病毒表达突变的S蛋白，这些突变蛋白包含来自世界各地的90多个临床分离株中发现的氨基酸替代。这些研究有望确定HmAbs在人类中作为被动免疫疗法对抗SARS冠状病毒感染的潜力。

Characterization of Therapeutic human monoclonal antibodies against SARS The discovery that a corona virus is the causative agent of SARS and its rapid genomic sequencing has created opportunities to develop effective modalities of prevention and treatment against this virus. Of particular interest is the spike glycoprotein (S protein), which is expressed as a surface projection. The S protein is involved in viral attachment, cell-cell fusion, the induction of protective immune response in virus neutralization. This proposal will focus on developing human monoclonal antibodies that can be used for prophylaxis, and/or post-exposure treatment of SARS-CoV infection. We have already generated a panel of hybridomas capable of producing neutralizing human monoclonal antibodies (HmAbs) in close collaboration with Abgenix Inc using their XenoMouse. These hybridomas have been cloned and 19 HmAbs have been produced, purified and re-tested in a neutralization assay. In addition, we have already produced several recombinant fragments of the S protein and obtained overlapping peptides that span the S protein, and used them to determine binding specificities of HmAbs. In aim-1 we will further characterize the neutralizing ability of human monoclonal antibodies against SARS-CoV in vitro and determine their relative binding affinities and specificities using a number of different current approaches. In aim-2, we will identify human monoclonal antibodies that can potentially neutralize a broad range of clinical isolates by testing them in a viral entry and a cell fusion assay using different pseudotyped viruses expressing mutant S proteins that contain amino acid substitutions identified in over 90 clinical isolates from around the world. These studies are expected to result in the characterization of HmAbs with potential as passive immunotherapeutics against SARS-CoV infection in humans.