人巨细胞病毒自然感染过程中体液免疫应答机制研究

Human cytomegalovirus (HCMV) is capable of latent infection in humans, which causes serious and even life-threatening disease in human populations with immune suppression or immunocompromised patients. . Due to the lack of animal model and full antigens, the neutralizing antibodies induced by HCMV and predominant neutralizing epitopes on HCMV antigen remain elusive. As a result, there have been several experimental vaccines which are difficult to induce potent neutralization failed in human clinical trials. Understanding the humoral immune response to human cytomegalovirus during natural infection and the characterization of dominant neutralizing epitopes on HCMV antigen need to be addressed urgently.. In previous study, with efficient method on human antibody isolation and PGM deep sequencing, we have established a panel of 56 HCMV-specific monoclonal antibodies from natural infected healthy donors, most of which exhibited potent neutralizing activity. In this study, we will establish a larger panel of HCMV-specific monoclonal antibodies from six healthy subjects. Using these antibodies as probes, we will discover the relationship between antigen specificity and antibody function, and further identify the dominant neutralizing epitopes precisely. To investigate the somatic variants driven by antigen, we will analyze the correlation between the size and complexity of antibody lineages and their antiviral function. Our study provides extensive understanding on active evolution and mechanism of humoral response against HCMV. Furthermore, the lineage character of dominant neutralizing antibodies and the distribution of corresponding epitopes on HCMV antigen may play an important role for the development of therapeutic antibody and prophylaxis vaccine.

人巨细胞病毒（HCMV）在人群中普遍感染且潜伏多年，机体免疫功能低下人群的新发或复发感染将引起致命疾病。由于缺乏动物模型及完整病毒蛋白抗原，HCMV诱导的中和抗体及病毒优势中和表位尚不清晰，多种疫苗的研制在临床实验中宣告失败。HCMV病毒自然感染者的体液免疫应答及病毒表面的优势中和表位尚待深入研究。在前期工作中，课题组建立了高效全人抗体筛选和PGM深度测序平台，从HCMV自然感染者中获得56株全人抗体，包含多株优势中和抗体。本研究将在此基础上获得6例HCMV自然感染者的特异全人抗体盘，揭示抗体的分子特征以及抗体功能与基因型的关系，鉴定出HCMV表面的优势中和表位，揭示抗原驱动下的抗体体细胞突变，分析突变抗体谱系复杂度与抗病毒功能的关系。本研究将深入认识HCMV自然感染过程中的体液免疫应答机制，获得的优势中和抗体谱系特征和识别的中和表位分布，对于HCMV治疗性抗体和疫苗的研发具有重要意义。

为了得到有效的防治型疫苗并开发治疗性中和抗体，了解HCMV自然感染人群的体液免疫效应以及探索天然感染病毒表面的中和优势表位就显得尤为重要。本研究结果深入探讨了HCMV潜在或持续性感染下的体液免疫应答机制，获得的优势中和抗体的谱系特征和识别的中和表位分布，可为HCMV治疗性抗体和疫苗的研发提供坚实的实验依据。具体结果如下：.1）建立HCMV特异性全人源抗体盘，获得多个优势中和抗体。发现HCMV病毒诱导强有力的中和活性与gH五聚体表位有关。大部分gB特异性抗体的病毒结合能力较强，与gB抗原为病毒表面最为丰富的蛋白之一有关。部分gB中和抗体具有补体依赖性。.2）揭示抗体的分子特征以及抗体功能与基因的关系，发现中和抗体的重链优势基因型在不同的志愿者中各不相同，Kappa链抗体在抗原结合反应中表现较优异，但在中和反应中lambda抗体的中和活性较强。中和抗体HCDR3区长度均明显偏长，在可变区的高度频变有利于产生有效的保护性抗体。gB蛋白较易产生基因型多样化的抗体，而gH抗体的基因相对保守。将志愿者的全人抗体各自建立基因进化树，发现大部分符合抗体结构与功能之间的SAR关系。.3）对HCMV全人抗体的进化发展进行探索，初步确立高亲和力的中和或结合抗体常常位于亲和力成熟过程中进化靠前且较为成熟的支系。高亲和力中和抗体家族为应对病毒感染的变化，通过重链体细胞频变过程为机体提供有效的动态保护作用。.4）鉴定出HCMV五聚体膜蛋白的8个抗原位点。证明与6个保守的免疫原性位点特异性抗体的竞争，可以作为评估疫苗免疫血清在中和潜在遗传多样性病毒株能力。