Identification and Characterization of Neutralizing Nanobodies Against Dengue Virus
Abstract
The Dengue virus (DENV) belongs to the flavivirus genus, Flaviviridae family. The mosquito-borne virus infects over 50 million people worldwide annually. Life-threatening conditions such as Dengue hemorrhagic fever and Dengue shock syndrome have been observed in DENV infected patients. The flavivirus envelope (E) protein has been recognized as the key target protein for antibody and vaccination studies because numerous neutralizing antibodies have been found to have preventative effects in animal models infected with DENV. However, because DENV has four serotypes that lead to Antibody-Dependent Enhancement (ADE) and a more severe disease upon second infections, development of effective DENV vaccines has been unsuccessful. In addition, due to the ADE effect, the neutralizing antibodies are not ideal therapeutics for DENV. Therefore, employed Nanobodies (Nbs) that are the smallest intact antigen-binding fragment of an antibody to overcome the ADE. Nbs lack the Fc domain in antibodies which would otherwise lead to ADE in antibodies. To identify neutralizing Nbs, the full-length ecto-domain and the domain 3 of the DENV E protein were recombinantly expressed bacteria and refold. E proteins were further used to screen the potential neutralizing Nbs from a commercial phage display library. Then an enzyme-linked immunosorbent assay (ELISA) was used to identify and confirm Nbs that could bind the DENV E proteins. Finally, the identified Nbs were cloned, expressed, and purified. The resulting Nbs were then evaluated for their neutralizing potency in a cell-based plaque reduction assay. These studies have the potential to contribute to antiviral treatments and prevention of DENV.