Optimization of recombinant Zika virus NS1 protein secretion from HEK293 cells

CASSOLA A; CASTILLO DS; ROLDAN, JS

Mar del Plata

Congreso; Reunión Anual de SOCIEDADES DE BIOCIENCIA; 2019

Zika virus (ZIKV) is a mosquito-borne flavivirus that has gained global attention after the 2015 outbreak in Brazil, where devastating congenital neurodevelopmental defects were associated with maternal ZIKV infection. Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect ZIKV acute infection to improve patient care and to control future outbreaks. Nonstructural 1 (NS1) glycoprotein results in an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient´s bloodstream early in the course of the infection. The aim of this study consisted of establishing a stable and optimized recombinant ZIKV NS1 (rZNS1) mammalian expression system in order to purify the hexameric protein, which can potentially be used in the development of diagnostic tests. Stable rZNS1-His-expressing HEK293 cells were generated through lentiviral transduction followed by dilution cloning, obtaining 1E4-C9 clone, which presented the highest intracellular and secreted rZNS1-His protein levels by Western blot assays. Optimization of rZNS1-His protein secretion in 1E4-C9 HEK293 cells was accomplished with 50 nM rapamycin treatment followed by serum-free media incubation for 9 days. Nickel affinity-purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient´s serum by indirect ELISA (iELISA) and Dot blot assays, and showed the ability to induce a humoral response in immunized mice as assessed by iELISA. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.