Tu Zhui, Huang Xiaoqiang, Fu Jinheng, Hu Na, Zheng Wei, Li Yanping, Zhang Yang
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China.
Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA.
Immunology. 2020 Sep;161(1):53-65. doi: 10.1111/imm.13224. Epub 2020 Jun 30.
Heavy-chain-only antibodies (HCAbs), which are devoid of light chains, have been found naturally occurring in various species including camelids and cartilaginous fish. Because of their high thermostability, refoldability and capacity for cell permeation, the variable regions of the heavy chain of HCAbs (VHHs) have been widely used in diagnosis, bio-imaging, food safety and therapeutics. Most immunogenetic and functional studies of HCAbs are based on case studies or a limited number of low-throughput sequencing data. A complete picture derived from more abundant high-throughput sequencing (HTS) data can help us gain deeper insights. We cloned and sequenced the full-length coding region of VHHs in Alpaca (Vicugna pacos) via HTS in this study. A new pipeline was developed to conduct an in-depth analysis of the HCAb repertoires. Various critical features, including the length distribution of complementarity-determining region 3 (CDR3), V(D)J usage, VJ pairing, germline-specific mutation rate and germline-specific scoring profiles (GSSPs), were systematically characterized. The quantitative data show that V(D)J usage and VHH recombination are highly biased. Interestingly, we found that the average CDR3 length of classical VHHs is longer than that of non-classical ones, whereas the mutation rates are similar in both kinds of VHHs. Finally, GSSPs were built to quantitatively describe and compare sequences that originate from each VJ pair. Overall, this study presents a comprehensive landscape of the HCAb repertoire, which can provide useful guidance for the modeling of somatic hypermutation and the design of novel functional VHHs or VHH repertoires via evolutionary profiles.
仅重链抗体（HCAbs）不含轻链，已在包括骆驼科动物和软骨鱼在内的多种物种中天然存在。由于其高热稳定性、可重折叠性和细胞渗透能力，HCAbs重链的可变区（VHHs）已广泛应用于诊断、生物成像、食品安全和治疗领域。大多数关于HCAbs的免疫遗传学和功能研究基于案例研究或有限数量的低通量测序数据。来自更丰富的高通量测序（HTS）数据的完整图景有助于我们获得更深入的见解。在本研究中，我们通过HTS克隆并测序了羊驼（小羊驼）中VHHs的全长编码区。开发了一种新的流程来对HCAbs库进行深入分析。系统地表征了各种关键特征，包括互补决定区３（CDR3）的长度分布、V（D）J使用情况、VJ配对、种系特异性突变率和种系特异性评分谱（GSSPs）。定量数据表明，V（D）J使用情况和VHH重组存在高度偏差。有趣的是，我们发现经典VHHs的平均CDR3长度比非经典VHHs的长，而两种VHHs的突变率相似。最后，构建了GSSPs以定量描述和比较源自每个VJ对序列。总体而言，本研究展示了HCAbs库的全面图景，可为体细胞超突变建模以及通过进化谱设计新型功能性VHHs或VHH库提供有用指导。
