Antigens co-encapsulated with protease inhibitor (U-Omp19) in polymeric nanoparticles

PRADO, LINEIA; PASQUEVICH KARINA; BRUNO LAURA; GUAIMAS, FRANCISCO; CORIA, LORENA; CASSATARO JULIANA

San Luis

Congreso; Reunión Anual de la Sociedad Argentina de Inmunología 2023; 2023

Vaccination continues to be one of the most important tools for the prevention of infectious diseases. The oral pathway of immunization is usually considered to present advantages such as easy application, low costs and development of local and systemic immunity. Regarding the few oral vaccines in the market most are inactivated or attenuated. The development of oral subunit vaccines has added obstacles to surmount such as, the hostile environment and the dilution suffered by the vaccine components in the intestinal trackt, making it difficult for the antigen and adjuvant to arrive with integrity to the inductive sites in the mucosa. In our laboratory, we have shown that the protease inhibitor U-Omp19 from Brucella spp. is an oral vaccine adjuvant that protects co-delivered antigens from proteolysis in the gastrointestinal tract and increases antigen specific adaptive immune responses. To our knowledge there are no other reports describing the use of protease inhibitors from bacteria as vaccine adjuvants. This project seeks to increase the protection of the antigen, by encapsulating it with U Omp 19 in polymeric nanoparticles. The method chosen for the synthesis of the nanoparticles was double emulsion- solvent evaporation (DE-SE). Encapsulation efficiency of each protein was determined with electrophoresis SDS-PAGE and antigen specific ELISAS. Characterization was done using DLS, SEM and z-potential. Cytotoxicity was measured using propidium yodide (IP) and internalization of the antigen was evaluated using flow cytometry and Confocal microscopy respectively. In both cases HT29 and Caco-2 cell lines where used. Encapsulation efficiency was found to be between 20-60%. The nanoparticles presented a hydrodynamic diameter between 328 and 744 nm, a surface charge between -8,2 mV and -32 mV and a PDI of 0,23-0,29. Interestingly, an increase in the internalization of the encapsulated antigen was observed compared with the non-encapsulated antigen using two different cell lines. Moreover, the nanoparticles exerted a cytotoxicity between 10-13 % in these cell lines. In conclusion, we developed polymeric nanoparticles that encapsulates both the antigen and the adjuvant and as a potential tool to increase antigen protection that resulted in higher amounts of antigen internalized in cell lines that mimic the intestinal barrier. Together these properties make these antigen-adjuvant containing nanoparticles suitable candidates for potential oral immunization, since an increase in antigen protection may be translated into an increase in the amount of antigen and adjuvant that reaches the inductive sites in the intestinal mucosa.