In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15–19 aa and 232–237 aa, are involved in binding with neutralizing anti-p35 antibodies.

在这项研究中，从人单链可变片段（scFv）的痘苗病毒（VACV）-免疫噬菌体展示文库中选择了五种针对菌落病毒（ECTV）的噬菌体展示抗体（pdAbs）。ELISA法表明，选定的pdAb可以识别ECTV，VACV和牛痘病毒（CPXV）。原子力显微镜观察了pdAb与VACV的结合。在噬菌斑减少中和测试中，选定的三株pdAb中和了天花病毒（VARV）。对ECTV，VARV，VACV和CPXV蛋白的蛋白质印迹分析表明，中和性pdAb与正痘病毒35 kDa蛋白结合，后者由VACV中与ORF H3L同源的开放阅读框编码。完整的人类抗体fh1A是在pdAb的VH和VL结构域的基础上构建的，证实了天花病毒，VACV和CPXV感染后斑块形成的剂量依赖性抑制作用。为了确定负责与中和pdAb结合的p35区域，设计了一组截短的p35蛋白并在其中表达鉴定出大肠杆菌细胞，以及被选定的中和性pdAb识别的最小p35片段。另外，应用肽噬菌体展示组合文库来定位表位。获得的数据表明，负责中和pdAb识别的表位是不连续的，位于两个p35区域（15-19个氨基酸和232-237个氨基酸）内的氨基酸残基与中和性抗p35抗体结合。