Production of soluble recombinant PEDV spike protein and validation of its antigenicity as a subunit vaccine

Abstract

Porcine epidemic diarrhea (PED) is a highly infectious disease occurring in 1- to 2-week-old piglets. Symptoms include watery diarrhea and dehydration. PED is also a serious economic problem due to the high mortality rate (ZL Li. et al., 2012). In 2013, a PED outbreak in the United States caused serious economic loss. Of late, PED has been reported in Asian countries as well. Therefore, development of an effective PED vaccine is the need of the hour. Presented here is a 2-part study, wherein we developed a subunit oral vaccine consisting of the S1(33-591) domain of the porcine epidemic diarrhea virus (PEDV) spike protein along with an M-cell targeting ligand to effectively stimulate a mucosal immune response. We selected the E. coli(BL21)DE3, pLysS host system as it is cost effective, capable of rapid growth, and is easily mass-produced. However, the spike protein linked to the M-cell targeting ligand was expressed as an inclusion body. To overcome this challenge, we employed a two-step strategy in Part 1. The first step involved solubilization to increase protein flexibility, while the second included renaturation, which helped in accurate protein folding for native activity. However, solubilization and renaturation of inclusion bodies often results in variable results. Therefore, we optimized the process to improve efficiency. The solubilization step was optimized by titrating urea concentration and pH, whereas renaturation of solubilized protein is comparable to flash dilution and pulsatile dilution. Each method has its pros and cons. Finally, we performed purification of the protein using anion exchange chromatography, optimized by adjusting the pH. Since the secondary structure of the protein is linked to its bioactivity, the secondary structure of the refolded protein was characterized in Part 2, using circular dichroism and Fourier transform infrared spectroscopy analysis. Finally, antigenicity of soluble recombinant PEDV spike protein was evaluated in vivo. We analyzed the levels of serum IgG and its subtype. Thus, solubilization and renaturation provide a method to utilize inclusion bodies to prepare a subunit vaccine.