Generation of a Nanobody Targeting the Paraflagellar Rod Protein of Trypanosomes

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.锥虫（Trypanosomes）是一类可感染人类与家畜并引发疾病的原生动物寄生虫，目前尚无可用疫苗。消除这类疾病亟需高灵敏度的诊断工具与高效的治疗策略。基于抗原检测的免疫诊断（immunodiagnostics）优于抗体检测，因为后者无法区分现症感染与既往治愈状态。经典单克隆抗体存在诸多局限：无法识别隐蔽表位（cryptic epitopes，因其分子量约150 kDa）、生产成本高昂，且需要冷链（cold chain）维护——而在大多为农村地区的锥虫病流行区，这一条件难以实现。纳米抗体（Nanobodies）是源自骆驼科动物重链抗体的重组、热稳定、小分子量（15 kDa）抗原特异性单域可变片段。相较于经典抗体，纳米抗体具备诸多优势，因此本研究探究了纳米抗体用于靶向锥虫特异性抗原的潜力及其诊断应用价值。本研究使用伊氏锥虫（Trypanosoma evansi）裂解液免疫一只羊驼。通过噬菌体展示（phage display）与生物淘选（bio-panning）技术，筛选得到一株可与所有受试锥虫物种及分离株发生交叉反应的纳米抗体Nb392。结合免疫印迹（immunoblotting）、免疫荧光显微镜术、免疫沉淀（immunoprecipitation）与质谱分析法（mass spectrometry），研究人员对该纳米抗体识别的靶标进行了鉴定。结果显示，Nb392可靶向伊氏锥虫、布氏锥虫（T. brucei）、刚果锥虫（T. congolense）以及间日锥虫（T. vivax）的鞭毛旁杆蛋白PFR1。研究人员使用两株鞭毛旁杆组装缺陷的RNA干扰突变体（PFR2RNAi与KIF9BRNAi）验证了该纳米抗体的特异性。综上，本研究通过使用复杂蛋白混合物免疫羊驼，成功制备得到一株高度特异性的纳米抗体Nb392，其可靶向锥虫鞭毛中的保守蛋白PFR1。Nb392可作为鞭毛旁杆的优质标记物，在锥虫病诊断中具有应用价值。此外，如本研究所示，Nb392还可作为研究工具或用于PFR蛋白的分离纯化。