Abstract
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.
Highlights
Antibody engineering has allowed for the development of many forms of antibodies for diagnostic and therapeutic use in recent decades [1]
Given the properties presented by camelid VHHs, the characteristics of the hantavirus nucleoprotein and the necessity of developing high-throughput, accurate, rapid, reliable, low-cost diagnostic methods for hantavirus infections, as well as the need for alternative approaches to prevent or control the course of the disease, our study aimed to identify VHHs capable of recognizing the prND85 of Araucaria hantavirus
Particular characteristics presented by the single domains (VHH) of camelid heavy chain antibodies have stimulated researchers to develop studies aiming to use these fragments as components of diagnostic devices or in anti-viral therapy [9], [11], [12], [42,43,44,45,46]
Summary
Antibody engineering has allowed for the development of many forms of antibodies for diagnostic and therapeutic use in recent decades [1]. In addition to conventional antibodies, camelids produce functional immunoglobulins composed only of heavy chains in which the antigen binding site is formed only by the single Nterminal variable domain, referred to as VHH [5], [6], [7]. With an approximate molecular weight of 15 kDa, VHH fragments are one-tenth the size of whole antibodies [3], [8]. Their small size, along with their ability to recognize weakly antigenic epitopes or epitopes that are inaccessible to conventional antibodies, their high solubility, thermal and pH stability, ability to cross dense tissues and lower production costs make VHHs versatile tools for biotechnological applications [3], [9], [10], [11]. VHHs have been applied in cell imaging studies, in vivo imaging of tumor tissue and to diagnose viral infections [4], [18], [19]
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