Phospha light kit

Female C57BL/6 mice were vaccinated with 10⁶ live or necrotic NS3 DC2.4 s.c. (standard prime-protocol) and challenged 2 weeks later by hydrodynamic injection of 20 μg of pNFS plasmid in TransIT-QR HD delivery solution (Mirus) in a volume of 1/10 body weight. Unvaccinated mice were challenged as a control. Hydrodynamic injection was performed as we described (Yu et al., 2014 (link)). In the pNFS plasmid used to challenge mice, NS3/4A protein expression is controlled by the mouse albumin promoter/α-fetoprotein enhancer ensuring hepatocyte-specific expression of HCV NS3/4A. Introns 1 and 2 were included to optimize expression. Secreted alkaline phosphatase (SEAP) is encoded as a fusion protein, preceded by the FMDV2A protease which is designed to self-cleave and release SEAP co-translationally. Any mouse which failed to receive the full challenge volume was excluded from further analysis. Serum samples were collected from the challenged mice at different time-points and levels of SEAP were measured using the Phospha-light kit (Applied Biosystems) in 96 well flat bottom white microplates (GreinerBioOne; Yu et al., 2014 (link)).

Overnight, Huh7-J20 reporter cells grown in a 96-well tissue culture plate were used to check the antiviral activity of the compounds using three different models of infection according to Magri et al. (2016) [42 (link)]. Huh7-J20 cells were pre-treated with different concentrations of the compounds for 1 h and were then infected in the presence of the compounds for 3 h. Next, the virus was removed, and fresh medium without compounds was added for 72 h (Model 1). In Model 2, all steps were the same as in Model 1; however after viral infection, fresh medium together with different concentrations of the compounds was added for 72 h. In Model 3, the various concentrations of the compounds were added only 3 h post infection for 72 h. The antiviral activity was determined by measuring the SEAP activity using the Phospha-Light kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions with some modifications. In brief, 50 µL of culture supernatant was mixed together with 50 µL of assay buffer and incubated for 7 min at room temperature. Next, 50 µL of freshly prepared chemiluminescent substrate was added and incubated in the dark for 45 min, and the SEAP level was measured using a luminometer.

mSEAP was quantified in tissue lysates using the Phospha-Light Kit (Applied Biosystems) following the manufacturer’s instructions and was normalized by the total protein concentration measured using the Pierce BCA Protein Assay (Thermo Fisher Scientific).