Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.呼吸道合胞病毒（Respiratory syncytial virus, RSV）是一种重要的人类病原体，绝大多数儿童在两岁前便会受到其感染。RSV融合（F）蛋白是人类抗体的主要靶标，其包含多个可诱导中和抗体产生的抗原区域。抗原位点IV在RSV F蛋白的预融合与后融合构象中均保持保守。已有研究表明，针对抗原位点IV的抗体能够结合并中和呼吸道合胞病毒与人偏肺病毒（human metapneumovirus, hMPV）。为探究抗原位点IV处的结合模式多样性，我们构建了一组共4株全新的人源单克隆抗体（monoclonal antibodies, mAbs），竞争结合实验结果显示这些抗体均结合于抗原位点IV。诱变实验结果显示，两株单克隆抗体（3M3与6F18）的结合与中和活性依赖于精氨酸（arginine, R）残基R429。我们在该位点发现了两株不依赖R429的单克隆抗体（17E10与2N6），它们能够中和RSV R429A突变株；其中一株抗体（17E10）可同时中和RSV与人偏肺病毒。为阐明交叉反应的作用机制，我们开展了竞争结合实验、重组蛋白诱变实验、肽段结合实验以及电子显微镜实验。研究发现，具有交叉反应活性的人源单克隆抗体17E10与RSV F蛋白的结合姿势与101F相似，这暗示其可能与hMPV F蛋白存在交叉反应。本研究提供的实验数据为RSV的免疫识别与疫苗设计提供了全新的研究思路，我们提出了结合姿势可影响单克隆抗体在RSV与人偏肺病毒间交叉反应活性的新颖观点。对人类抗体结合的位点IV表位进行表征，可为泛肺病毒疫苗的设计提供参考依据。