Plaque reduction neutralization test

SdAb were evaluated for neutralizing activity against VEEV (strain TC-83) and CHIKV (strain 181/25) in Vero cells. Two-fold serial dilutions of each sdAb were prepared starting at a concentration of 20 μg/mL. Dilutions were incubated with virus (~ 150–300 plaque forming units, PFU) overnight at 4 °C. Dilutions were plated in duplicate wells of a 6-well plate of Vero cells for 1.5 ± 0.5 h with gentle rocking every 15 min. Cells were covered with 0.6% agarose overlay in 1 × BME (Thermofisher) to each well and incubating for 17 h for TC-83 and 24 h for CHIKV at 37 °C in a humidified 5% CO₂ atmosphere before the addition of the second 0.6% agarose overlay supplemented with neutral red vital stain. The plates were incubated at 37 °C for another 22–24 h before the transparent plaques were counted. The average of the duplicates was used to calculate 50% and 80% plaque reduction neutralization titer (PRNT₅₀ and PRNT₈₀) of each sdAb using the Matlab Curve Fitting tool. Values were determined by piecemeal interpolation with either a linear or exponential function, depending on the shape of the curve at the relevant point. When appropriate, the starting concentration of the sdAb construct was adjusted to include the dilutions ranging between PRNT₅₀ and PRNT₈₀. The average and standard deviation (STDEV) listed in the table were obtained from biological replicates. A similar method was used to determine the ability of standard and bivalent sdAb to neutralize wild type VEEV (strain TrD) and EEEV strain FL93-939³¹. The detailed protocol used to determine the ability of the sdAb to neutralize WEEV Imperial 181 strain³² (a gift from Dr. Aaron Brault) is presented in the supplemental material.