Table_3_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（envelope glycoprotein 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HCV疫苗研发的核心目标。然而，介导广谱中和作用的关键抗体分子特征仍未明确。 方法 为鉴定与广谱中和作用相关的B细胞库（B cell repertoire）特征，我们对HCV感染个体中HCV E2反应性B细胞的B细胞受体（B cell receptor, BCR）进行了RNA测序，这些受试者的血浆中和广度可分为高、低两个类别。随后，我们通过配对自发清除感染、高中和活性受试者中鉴定出的公共克隆型内丰度最高的重链与轻链，成功制备了单克隆抗体（monoclonal antibody, mAb）。 结果 我们发现了与HCV广谱中和作用相关的独特BCR特征，包括较长的重链互补决定区3（complementarity determining region 3, CDRH3）、特定的VH基因使用偏好、升高的体细胞超突变频率以及特异性VH基因突变。最引人关注的是，我们鉴定出多种仅存在于产生高中和活性血浆的受试者中的E2反应性公共BCR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）。其中大部分公共克隆型可被两名自发清除感染的受试者共享。从这些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所制备的单克隆抗体，富集了高中和克隆型的典型特征，如更高的体细胞超突变频率与IGHV1-69基因使用偏好，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表征，可为HCV疫苗研发提供重要的理论参考与研发方向。