2023年3月24日，上海市免疫学研究所叶菱秀团队在Science Immunology期刊在线发表了题为“DNA Repair Mechanisms That Promote Insertion-Deletion Events During Immunoglobulin Gene Diversification”的研究论文。该论文鉴定了在抗体多样化过程中低频率DNA片段插入事件和±1bp缺失或插入事件，揭示了±1bp和>1bp缺失或插入产生机制的差异。在此基础上，通过外切酶或聚合酶的遗传学操作获得了能够增加抗体基因缺失或插入的小鼠模型，为广谱中和抗体的筛选提供了新的思路和方法。
DNA片段缺失或插入是基因组中单个或多个核苷酸的缺失或插入。在免疫球蛋白即抗体的基因可变区外显子中，片段缺失或插入(<50 bp)通常会导致B细胞受体编码错误。然而，“塞翁失马，焉知祸福”，长片段缺失或插入又是罕见抗病毒广谱中和抗体(broadly neutralizing antibody, bnAbs)获得广谱中和能力的关键。例如，发生在抗HIV广谱中和抗体基因上的片段插入大大提高了抗体与HIV包膜三聚体表位的结合力。但是，由于片段缺失或插入在抗体多样化过程中发生频率较低，很难用传统模型和方法检测得到。因此，片段缺失或插入在抗体多样化中的分子机制仍知之甚少。本研究利用一种抗体无功能等位基因系统，同时结合超深度测序克服了片段缺失或插入检测的困难，成功捕捉到生理条件下真实的片段缺失或插入事件（图1）。通过对片段缺失或插入图谱的分析发现±1bp缺失或插入发生最频繁，占总缺失或插入事件的45%左右（图2）。对>1bp碱基插入的序列特征进行分析发现大部分的碱基插入为邻近序列的复制插入。发生在CDR3上的复制插入能够增加CDR3的长度，可能促进广谱中和抗体的产生。
图1 小鼠模型与实验设计流程
Schematic of the study design.
图2 抗体无功能等位基因上1bp和>1bp缺失或插入的占比
1 bp indels events are prevalent.
在体细胞高频突变（Somatic Hypermutation, SHM）过程中，胞苷脱氨酶AID能够作用于抗体可变区基因，引起DNA损伤。该损伤可以通过碱基切除修复途径（base excision repair, BER）或错配修复途径（mismatch repair, MMR）进行修复，产生碱基突变以及DNA片段的缺失或插入。为了探究DNA片段缺失或插入产生的分子机制，本研究在抗体无功能等位基因小鼠模型基础上构建了15种具有代表性的DNA损伤应答修复因子缺陷小鼠模型（图1），并对其片段缺失或插入图谱进行了系统的描绘（图3）。研究发现±1bp与>1bp缺失或插入产生的分子机制截然不同（图3-4），1bp缺失通过BER途径产生，1bp插入既可以通过BER途径产生，也可以通过MMR途径产生，产生过程需要核酸外切酶ExoI和易错DNA聚合酶Polη的参与。>1bp缺失或插入都是通过BER途径产生的，并且都需要DNA双链断裂损伤修复因子53BP1的参与。此外，>1bp插入还需要非同源末端连接途径和DNA聚合酶Polλ的参与。对片段缺失或插入分子机制的全面解析有助于理解广谱中和抗体产生的机制，为广谱中和抗体的生产提供了新的思路，为HIV等疫苗的设计提供了新的理论基础。
图3：抗体基因SHM中DNA片段缺失或插入机制的系统解析
Summary of the AID-initiated mutagenic outcomes of the different genetic models used in this study.
在机制研究基础上，本研究成功构建了能够增加抗体基因缺失或插入频率的小鼠模型（图3），核酸外切酶Trex2过表达的小鼠模型能够促进片段缺失的产生，DNA聚合酶Polβ条件性敲除的小鼠模型能够促进片段插入的产生。这两种小鼠模型可以与人源化抗体小鼠模型联合使用，用于疫苗的研发和广谱中和抗体的生产。
图4：抗体基因SHM中DNA片段缺失或插入机制的系统解析
Distinct factors/pathway that generate the 1-bp or longer indel events.
304am永利集团官网上海市免疫学研究所叶菱秀研究员，瑞典卡罗琳斯卡304am永利集团官网Qiang Pan-Hammarström教授和意大利都灵大学病理学系Roberto Chiarle教授为本论文的共同通讯作者，郝茜助理研究员和占传棕博士研究生为本论文的共同第一作者。本研究得到了国家重点研发计划、国家自然科学基金、304am永利集团官网国际科研合作项目以及中国博士后科学基金等资助。我们衷心感谢所有参与本研究的合作团队成员，同时感谢304am永利集团官网基础304am永利集团官网和上海市免疫学研究所的公共技术平台以及免疫所与瑞金医院共建的免疫与疾病研究中心对本研究的大力支持。
叶菱秀，研究员，博士生导师。2010年博士毕业于剑桥大学， 2010～2015年于哈佛大学304am永利集团官网进行博士后研究，2016年加入上海交通大304am永利集团官网上海市免疫学研究所并担任抗体多样化课题组长。获得国家自然科学基金优秀青年项目、国际合作项目等基金资助。叶菱秀课题组长期致力于B淋巴细胞抗体多样化分子机制研究，特别是广谱中和抗体与自身抗体产生机制。课题组在体液免疫反应和中和抗体发现上积累了多种技术和完善的研究体系。实验室已培养“博新计划”博士后、博士研究生多名。欢迎对本课题组研究感兴趣的学生及博士后的加入，共同探讨推进B细胞抗体多样化研究的进展。课题组主页为/sii/info/1053/1455.htm，欢迎感兴趣的申请者来函咨询yeaplengsiew@shsmu.edu.cn。
Study in Science Immunology reveals the mechanisms that generate insertion and deletion events during antibody diversification
Researchers from the laboratory of Leng-Siew Yeap at the Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Qiang Pan-Hammarström at the Karolinska Institute and Roberto Chiarle at Harvard University and the University of Torino have reported the results of their work to elucidate the mechanisms that generate rare mutagenic outcomes of antibody diversification process, with implications for the generation of antiviral broadly neutralizing antibodies and the development of autoantibodies.
The study, published online on 24 March 2023 in Science Immunology, represents a major advance in understanding the mechanisms underlying the generation of insertions and deletions (indels), rare outcomes of a physiological process that generates mutations in DNA to produce good antibodies. Indels are generally undesirable in the genome, but they are common and have been shown to be functionally important in rare antibodies that can neutralize a wide range of viral strains. To study the mechanisms that generate insertions and deletions, the researchers take advantage of a powerful passenger-antibody gene knock-in mouse model, which allows the detection of unselected mutational events, and ultra-deep sequencing of a large numbers of germinal center B cells to gain insight into even relatively rare events. This led to the revelation that ±1 bp indel events, which are deleterious to the B cell receptor function, are the most common indel events, while longer indel events, which can generate productive antibodies, are rare. By analyzing a large number of DNA repair gene-deficient mice carrying the passenger allele, the researchers identified the different DNA repair pathways that generate indels of different sizes, e.g. the mechanisms that generate 1 bp events are not the same as those that generate longer indels. The study also generated mutant mouse models with either increased deletions or insertions, which could potentially be used to identify rare antibodies.
The co-first authors of this paper are Dr. Qian Hao, a research associate, and Chuanzhong Zhan, a PhD student in the Yeap Lab. This work was supported by funding from the National Key R&D Program of China, the National Natural Science Foundation of China, the NSFC-MAECI Italy-China Collaborative grant, the Joint Research Initiative of the School of Medicine, Shanghai Jiao Tong University, the Swedish Research Council, the Swedish Cancer Society, CIMED, the Alice Wallenberg Foundation, and the China Postdoctoral Science Foundation. We would like to thank all of our wonderful collaborators on this study who are not mentioned here individually. The authors also acknowledge the support of the Center for Immune-Related Diseases at Shanghai Institute of Immunology and Ruijin Hospital and Core Facilities at the Shanghai Institute of Immunology and the School of Basic Medical Science.
The Yeap lab welcomes enthusiastic students and postdoctoral fellows to join the group. Interested individuals can contact Dr. Yeap at yeaplengsiew@shsmu.edu.cn.
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