(331c) Desolvent-Free Vaccine Platform to Enhance Effectiveness of Protein Subunit Vaccines

Vaccination has provided the most effective protection against viruses. Subunit vaccines have significant advantages over whole killed or inactivated viruses in terms of immune specificity and safety. However, protein subunit vaccines can have challenges such as low immunogenicity. Previously, our lab overcame this challenge by development of protein nanoparticles, developed by desolvating whole protein antigens and stabilizing via covalent crosslinking. Protein nanoparticles have been made from several antigens, including the highly conserved domains of influenza proteins such as the stalk of hemagglutinin and nucleoprotein. While the particles induce protective humoral and cellular responses, the desolvent, typically an alcohol, compromised the structure of the antigen. It is particularly important to preserve conformational epitopes in protein nanoparticle antigens in order to engage B cell receptors directly and enhance the strength and breadth of the humoral response. We hypothesized that effectiveness of vaccine protein nanoparticles could be improved by developing desolvent free fabrication processes that maintain antigen structure. Desolvent-free nanoparticles were synthesized by using ovalbumin as a model antigen via different approaches. Directly crosslinking or salt precipitating ovalbumin were identified as the best methods in terms of physicochemical properties (size~ 250 nm and PDI~ 0.2-0.29). These methods were also confirmed to produce hemagglutinin particles, an antigen that was unfolded by the desolvation process. Retention of ovalbumin conformation in desolvent-free nanoparticles was observed by circular dichroism while conformation of ovalbumin in desolvated nanoparticles was significantly compromised. The same phenomenon was also confirmed by molecular dynamics simulation of ovalbumin folding in the alcohol desolvent mixture compared to water. To evaluate the effect of desolvent-free nanoparticles on immune system activation, dendritic cells were stimulated with desolvated and desolvent-free nanoparticles. Surprisingly, expression levels of CD86, a maturation marker of dendritic cells, elicited by desolvent-free nanoparticles were higher than those by desolvated nanoparticles. For in vivo efficacy evaluation of the desolvent-free vaccines, mice have been injected intramuscularly with desolvent-free and desolvated nanoparticles, and also coated nanoparticles which were prepared by crosslinking ovalbumin proteins to the surface of desolvated nanoparticles. While coated desolvated nanoparticles were previously shown to better elicit immune responses than uncoated desolvated nanoparticles, the coated desolvated nanoparticles can only incorporate a limited amount of antigens with preserved structure and have not been directly compared to the desolvent-free nanoparticles. The results from in vivo study in concert with in vitro study will be presented and will reveal whether the new desolvent-free platform can be a promising strategy to improve immune responses to subunit protein vaccines.