US Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, 4555 Overlook Ave SW, Washington, DC, 20375, USA.
Microb Cell Fact. 2017 Dec 12;16(1):223. doi: 10.1186/s12934-017-0837-z.
A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity.
An immune phage display library containing more than 10 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies.
SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.
重组抗体技术的一个关键优势是能够优化和定制试剂。单域抗体（sdAbs）是从骆驼科和鲨鱼重链抗体中重组产生的可变结构域，具有稳定性和溶解性的优势，并且可以进一步工程改造以增强其性能。在这项研究中，我们生成了针对埃博拉病毒包膜糖蛋白（GP）的 sdAbs，并提高了它们的稳定性，以扩大其在艰苦环境下的应用。埃博拉病毒具有极强的毒性，约 50%的病例会导致致命性出血热。病毒 GP 与宿主细胞受体结合，促进病毒进入，因此在致病性方面起着关键作用。
我们从用灭活的埃博拉病毒和重组产生的 GP 联合免疫的骆驼中开发了一个包含超过 10 个独特克隆的免疫噬菌体展示文库。我们对文库进行淘选以获得与 GP 结合的 sdAbs，并从 5 个不同的序列家族中分离出 sdAbs。选择了 3 种具有解离常数范围为 2-20 nM 和熔点范围为 57-72°C 的 GP 结合 sdAbs 进行蛋白质工程改造，通过共识序列突变和添加非典型二硫键的组合来提高其稳定性。这些变化使 sdAbs 的熔点提高了 15-17°C。此外，在 C 末端融合一个短的正电荷尾巴，为这些 sdAbs 的化学修饰提供了理想的位点，从而提高了 GP 和埃博拉病毒样颗粒的检测限，同时作为示踪抗体。
选择了针对埃博拉 GP 的 sdAbs，并利用蛋白质工程提高了它们的稳定性和功能。在缺乏可靠冷藏的艰苦环境中，抗体试剂的热稳定性可能尤为重要。未来的研究可以评估这些分离的 sdAbs 作为埃博拉诊断或治疗应用候选物的潜力。
