Table_4_Convergent antibody responses are associated with broad neutralization of hepatitis C virus.docx

IntroductionEarly development of broadly neutralizing antibodies (bNAbs) targeting the hepatitis C virus (HCV) envelope glycoprotein E2 is associated with spontaneous clearance of infection, so induction of bNAbs is a major goal of HCV vaccine development. However, the molecular antibody features important for broad neutralization are not known.
MethodsTo identify B cell repertoire features associated with broad neutralization, we performed RNA sequencing of the B cell receptors (BCRs) of HCV E2-reactive B cells of HCV-infected individuals with either high or low plasma neutralizing breadth. We then produced a monoclonal antibody (mAb) expressed by pairing the most abundant heavy and light chains from public clonotypes identified among clearance, high neutralization subjects.
ResultsWe found distinctive BCR features associated with broad neutralization of HCV, including long heavy chain complementarity determining region 3 (CDRH3) regions, specific VH gene usage, increased frequencies of somatic hypermutation, and particular VH gene mutations. Most intriguing, we identified many E2-reactive public BCR clonotypes (heavy and light chain clones with the same V and J-genes and identical CDR3 sequences) present only in subjects who produced highly neutralizing plasma. The majority of these public clonotypes were shared by two subjects who cleared infection. A mAb expressing the most abundant public heavy and light chains from these clearance, high neutralization subjects had features enriched in high neutralization clonotypes, such as increased somatic hypermutation frequency and usage of IGHV1-69, and was cross-neutralizing.
DiscussionTogether, these results demonstrate distinct BCR repertoires associated with high plasma neutralizing capacity. Further characterization of the molecular features and function of these antibodies can inform HCV vaccine development.

**引言** 靶向丙型肝炎病毒（hepatitis C virus, HCV）包膜糖蛋白E2的广谱中和抗体（broadly neutralizing antibodies, bNAbs）的早期产生与感染的自发清除密切相关，因此诱导广谱中和抗体是丙型肝炎病毒疫苗研发的核心目标之一。然而，介导广谱中和活性的关键抗体分子特征尚未明确。**方法** 为鉴定与广谱中和活性相关的B细胞库特征，我们对感染丙型肝炎病毒且血浆中和活性高低不同的个体中，靶向HCV E2的B细胞的B细胞受体（B cell receptors, BCRs）进行了RNA测序。随后，我们从自发清除感染且中和活性较高的受试者中鉴定得到的公共克隆型（public clonotypes）中，选取丰度最高的重链与轻链进行配对，制备了单克隆抗体（monoclonal antibody, mAb）。**结果** 我们发现了与HCV广谱中和活性相关的独特B细胞受体特征，包括较长的重链互补决定区3（heavy chain complementarity determining region 3, CDRH3）、特定的可变重链（VH）基因使用偏好、更高的体细胞超突变频率，以及特定的VH基因突变特征。最引人关注的是，我们鉴定出多种仅在产生高中和活性血浆的受试者体内存在的、靶向E2的公共B细胞受体克隆型（即拥有相同V、J基因且CDR3序列完全一致的重链与轻链克隆）。其中大多数公共克隆型可在两名自发清除感染的受试者间共享。从上述高中和活性且清除感染的受试者中选取丰度最高的公共重链与轻链制备的单克隆抗体，具备高中和活性克隆型所富集的特征，比如更高的体细胞超突变频率、IGHV1-69基因的使用偏好，且具备交叉中和活性。**讨论** 综上，本研究结果证实存在与高血浆中和能力相关的独特B细胞库。对这些抗体的分子特征与功能开展进一步解析，可为丙型肝炎病毒疫苗研发提供理论依据。