Proteolytic resistance of anti-VP6 VHH

In order to study the stability and protease resistance of the VHH we conducted two separated experiments in which we examined stability at low pH and resistance to gastric and intestinal enzymes for 2KD1 and 3B2, respectively. To study stability and resistance of the VHH in conditions that emulate the host’s stomach, aliquots consisting of 400 μg/ml of 2KD1 or 3B2 were incubated in Simulated Gastric Fluid (SGF; pH 1.2) with and without enzymes for up to one hour at 37°C. We also tested the resistance to gastric enzymes in the presence of ORS. The experiment included the following samples: 3B2+SGF (pH 1.2); 3B2+SGF without enzymes (pH 1.2); 3B2+SGF+ORS (pH 7.1). The SGF was prepared following the US Pharmacopeia guidelines, with a final porcine pepsin concentration of 0.32% w/v and pH 1.2. On the other hand, we examined the VHH’s resistance to intestinal proteases, by incubating 400 μg/ml of 2KD1 or 3B2 in Simulated Intestinal Fluid (SIF) with or without ORS for up to one hour at 37°C: 2KD1+SIF (pH 6.8); 2KD1+SIF without enzymes (pH 6.8); 2KD1+SIF+ORS (pH 7.1). The SIF was also prepared following the US Pharmacopeia guidelines, with a final pancreatine concentration of 1% w/v and pH 6.8. All samples were analyzed by an ELISA that measures VHH titers against RVA VP6 to determine the remaining percentage of functional VHH, as was previously described.