Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel–PspA formulation. First, to characterize the nanogel–PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methodsa densitometric method based on lithium dodecyl sulfate (LDS)–PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)to determine the PspA content in the nanogel formulations. Third, treatment of nanogel–PspA formulations by adding methyl-β-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel–PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel–PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS–PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS–PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel–PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.