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 methodsa 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.