Pereira Soraya S, Moreira-Dill Leandro S, Morais Michelle S S, Prado Nidiane D R, Barros Marcos L, Koishi Andrea C, Mazarrotto Giovanny A C A, Gonçalves Giselle M, Zuliani Juliana P, Calderon Leonardo A, Soares Andreimar M, Pereira da Silva Luiz H, Duarte dos Santos Claudia N, Fernandes Carla F C, Stabeli Rodrigo G
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, RO, Brazil.
Instituto Carlos Chagas, Fiocruz Paraná, Curitiba, PA, Brazil.
PLoS One. 2014 Sep 22;9(9):e108067. doi: 10.1371/journal.pone.0108067. eCollection 2014.
In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus infections.
除了传统抗体外，骆驼科动物还产生仅由重链组成的免疫球蛋白G，其中抗原结合位点仅由称为VHH的单个结构域形成。它们的特殊特性使VHH成为药物递送、被动免疫疗法和高通量诊断的有趣工具。汉坦病毒是布尼亚病毒科的啮齿动物传播病毒。已知该感染有两种临床形式。肾综合征出血热（HFRS）出现在旧世界，而汉坦病毒肺综合征（HPS）出现在美洲大陆。HPS没有特异性治疗方法，其诊断通过分子或血清学技术进行，主要使用单克隆抗体或汉坦病毒核蛋白（N）来检测患者血清中的IgM和IgG。本研究提出使用骆驼科动物VHH来开发诊断和确认HPS的替代方法。采用噬菌体展示技术获得VHH。用巴西汉坦病毒株的重组N蛋白（prNΔ₈₅）免疫一只羊驼后，分离VHH区域构建免疫文库。VHH与M13KO7噬菌体外壳蛋白III融合展示，并在固定化的prNΔ₈₅上进行筛选步骤。筛选后，80个克隆特异性识别N蛋白。对这些克隆进行测序，主要根据互补决定区进行分组，并通过蛋白质印迹（WB）、表面等离子体共振（SPR）装置和酶联免疫吸附测定（ELISA）对5个克隆进行分析。除了通过WB识别prNΔ85的能力外，所有选定克隆的亲和常数都在纳摩尔范围内。此外，克隆KC329705能够检测溶液中的prNΔ₈₅以及天然病毒抗原。研究结果支持这样的假设，即选定的VHH可能是开发快速准确的HPS诊断检测方法的有力工具，这对于为患者提供支持性护理和降低与汉坦病毒感染相关的高死亡率至关重要。
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