Antigenic Fingerprinting following Primary RSV Infection in Young Children Identifies Novel Antigenic Sites and Reveals Unlinked Evolution of Human Antibody Repertoires to Fusion and Attachment Glycoproteins

Respiratory Syncytial Virus (RSV) is the major cause of pneumonia among infants. Here we elucidated the antibody repertoire following primary RSV infection and traced its evolution through adolescence and adulthood. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes in the RSV fusion protein (F) and attachment protein (G) were used for unbiased epitope profiling of infant sera prior to and following RSV infection. F-GFPDL analyses demonstrated modest changes in the anti-F epitope repertoires post-RSV infection, while G-GFPDL analyses revealed 100-fold increase in number of bound phages. The G-reactive epitopes spanned the N- and C-terminus of the G ectodomain, along with increased reactivity to the central conserved domain (CCD). Panels of F and G antigenic sites were synthesized to evaluate sera from young children (<2 yr), adolescents (14–18 yr) and adults (30–45 yr) in SPR real-time kinetics assays. A steady increase in RSV-F epitope repertoires from young children to adults was observed using peptides and F proteins. Importantly, several novel epitopes were identified in pre-fusion F and an immunodominant epitope in the F-p27. In all age groups, antibody binding to pre-fusion F was 2–3 folds higher than to post-fusion form. For RSV-G, antibody responses were high following early RSV infection in children, but declined significantly in adults, using either G proteins or peptides. This study identified unlinked evolution of anti-F and anti G responses and supportive evidence for immune pressure driven evolution of RSV-G. These findings could help development of effective countermeasures including vaccines.

呼吸道合胞病毒（Respiratory Syncytial Virus, RSV）是引发婴幼儿肺炎的首要致病原。本研究阐明了初次感染RSV后的抗体谱特征，并追踪了该抗体谱在青春期至成年阶段的演化过程。我们采用在RSV融合蛋白（F）与附着蛋白（G）中展示线性及构象表位的全基因组片段噬菌体展示文库（Whole genome-fragment phage display libraries, GFPDL），对RSV感染前后的婴幼儿血清开展无偏倚表位谱分析。F-GFPDL分析结果显示，RSV感染后抗F表位谱仅出现小幅变化；而G-GFPDL分析则显示结合噬菌体的数量提升了100倍。与G蛋白反应的表位覆盖了G蛋白胞外域的N端与C端，同时对中央保守域（Central Conserved Domain, CCD）的反应性有所增强。我们合成了一系列F和G蛋白的抗原位点，利用表面等离子体共振（Surface Plasmon Resonance, SPR）实时动力学实验评估了幼儿（<2岁）、青少年（14~18岁）及成年人（30~45岁）的血清样本。通过肽段与F蛋白实验，我们观察到从幼儿到成年人的RSV-F表位谱呈现稳步上升趋势。尤为重要的是，我们在融合前构象的F蛋白中发现了多个全新表位，并在F-p27区域鉴定出一个免疫优势表位。在所有年龄组中，针对融合前F蛋白的抗体结合水平均较融合后F蛋白高出2~3倍。对于RSV-G而言，儿童在早期感染RSV后抗体应答水平较高，但无论是使用G蛋白还是肽段进行检测，成年人的该应答水平均出现显著下降。本研究揭示了抗F与抗G抗体应答的非协同演化过程，并为免疫压力驱动的RSV-G演化提供了佐证。本研究结果可为包括疫苗在内的有效防治手段的开发提供参考。