Empirical phase diagram approach toward biophysical characterization of vaccine candidates against Shigella, Salmonella and Yersinia

Abstract

Infectious diarrhea is an important public health problem and a major cause of morbidity all over the world. Infants and young children are the most vulnerable group. Vaccination is one of the most important public health tools for the prevention of infectious diseases, however, formulation of safe and effective vaccines against diarrheal disease can be challenging. Shigella, Salmonella and Yersinia enterocolitica are three highly virulent pathogens in urgent need of vaccine development. A common virulence factor is utilized by these three pathogens, the type III secretion system (T3SS), which is highly conserved across multiple serotypes within these groups. With the discovery that the Shigella T3SS proteins IpaD and IpaB are protective antigens, we constructed a novel IpaD-IpaB fusion protein to simplify the production and reduce the cost of vaccine production. Because of its hydrophobic IpaB portion, the DB fusion needs detergent to maintain solubility. A mild detergent called LDAO was identified and showed great promise for protein stabilization when compared to the detergent used previously (called OPOE). Inspired by the success of the DB fusion, we constructed a SipDB fusion using the homologous Salmonella T3SS proteins SipD and SipB. In addition to exploring the fusion strategy with regard to anti-Shigella and anti-Salmonella vaccines, a relatively new antigen delivery system called Bacterium-Like Particles (BLPs) was also explored and formulated as a potential means for delivering protective antigens from Shigella, Salmonella and Yersinia enterocolitica (LcrV and YopB). Derived from Lactococcus lactis, BLPs are peptidoglycan skeletons that are safe for newborns and can carry multiple antigens on their surface. The T3SS proteins were fused with a protein anchor domain for BLP attachment. The constructed fusion vaccine candidates and BLP-based vaccine candidates were biophysically characterized using multiple techniques including circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, and static light scattering which allowed measuring the secondary, tertiary and quaternary structural changes, respectively, as a function of environmental stress. The resulting large dataset was summarized using a three-index empirical phase diagram (EPD), which is a colored representation of the overall structural integrity and conformational stability of the vaccine candidates in response to environmental conditions. The information acquired is used for identifying favorable states of protein and the proper detergent to be used for the formulation of the resulting vaccines. This approach can also be used in further studies on excipient screening for stabilizing final vaccine products, though that work was not done here.