Department of Virology, Medicum, University of Helsinki, Helsinki, Finland.
Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki, Finland.
J Gen Virol. 2023 Oct;104(10). doi: 10.1099/jgv.0.001901.
Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the family of the order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope on the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthermore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60 % of these sites reside within or close to the epitope of mAb 4G2, indicating that this region may represent a crucial area targeted by neutralizing antibodies against PUUV, and to a lesser extent, other hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.
鉴定 B 细胞表位有助于疫苗、治疗性抗体和诊断工具的开发。此前，我们使用组合方法（包括肽扫描、噬菌体展示和水疱性口炎病毒（VSV）颗粒的定点突变）预测了针对普马拉病毒（PUUV，正布尼亚病毒科汉坦病毒属）的田鼠单克隆抗体（mAb）4G2 的结合位点。这些技术导致了中和逃逸突变 F915A 的鉴定。令我们惊讶的是，最近 PUUV Gc 与 Fab 4G2 复合物的晶体结构表明，残基 F915 远离 mAb 4G2 的表位。为了澄清这个问题并探讨不一致的潜在解释，我们设计了一个诱变实验来探测 4G2 表位，使用三个携带氨基酸变化 E725A、S944F 和 S946F 的 PUUV 假病毒，这些氨基酸变化位于基于结构的 4G2 表位上。这些氨基酸变化能够赋予对 4G2 的中和逃逸能力，并且 S944F 和 S946F 还赋予了对人 mAb 1C9 的中和逃逸能力。此外，我们将来自汉坦病毒 Gc 的所有已知中和逃逸位点映射到 PUUV Gc 上，结果表明，超过 60%的这些位点位于或靠近 mAb 4G2 的表位内或附近，这表明该区域可能代表针对 PUUV 的中和抗体的一个关键靶向区域，对其他汉坦病毒的靶向性则较弱。该脆弱性位点的确定可能为未来针对 PUUV 和其他汉坦病毒的亚单位疫苗的开发提供指导。
