Novel Camelid Antibody Fragments Targeting Recombinant Nucleoprotein of Araucaria hantavirus: A Prototype for an Early Diagnosis of Hantavirus Pulmonary Syndrome

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.除常规抗体外，骆驼科动物还可产生仅由重链构成的免疫球蛋白G（immunoglobulin G），其抗原结合位点仅由被称为VHH的单结构域形成。其独特特性使得VHH成为药物递送、被动免疫治疗以及高通量诊断领域极具应用价值的工具。汉坦病毒（Hantaviruses）是布尼亚病毒科（Bunyaviridae）的啮齿动物传播的病毒。目前已知该病毒感染存在两种临床分型：肾综合征出血热（Hemorrhagic Fever with Renal Syndrome, HFRS）流行于旧大陆，而汉坦病毒肺综合征（Hantavirus Pulmonary Syndrome, HPS）则见于美洲大陆。目前针对汉坦病毒肺综合征尚无特效治疗手段，其诊断主要依靠分子或血清学技术，通常利用单克隆抗体（monoclonal antibodies）或汉坦病毒核蛋白（N）来检测患者血清中的IgM与IgG抗体。本研究提出利用骆驼源VHH开发用于汉坦病毒肺综合征诊断与确认的替代方法。研究采用噬菌体展示技术（phage display technology）获取VHH：首先用巴西汉坦病毒毒株的重组核蛋白（prNΔ85）免疫一只大羊驼（Lama glama），随后分离VHH区域以构建免疫文库；将VHH与M13KO7噬菌体衣壳蛋白III（M13KO7 phage coat protein III）融合展示，并通过固定化的prNΔ85完成筛选步骤。筛选结束后，共获得80株可特异性识别核蛋白的克隆。对这些克隆进行测序，并主要依据互补决定区（CDRs）进行聚类，随后选取5株克隆进行蛋白质印迹（western blot, WB）、表面等离子体共振（surface plasmon resonance, SPR）检测以及酶联免疫吸附实验（ELISA）分析。除可通过蛋白质印迹识别prNΔ85外，所有筛选得到的克隆均展现出纳摩尔级别的亲和常数。此外，克隆KC329705还能够在溶液中识别prNΔ85以及天然病毒抗原。上述研究结果验证了这一假说：筛选得到的VHH可作为开发快速精准的汉坦病毒肺综合征诊断检测方法的有力工具，而此类检测方法对于为患者提供支持性治疗、降低汉坦病毒感染相关的高死亡率至关重要。