Hepatitis B virus causes acute and
chronic infections in millions of people worldwide and, since 1982, a
vaccine with 95%
effectiveness has been available for
immunization. The main component of the recombinant
hepatitis B vaccine is the
surface antigen protein (
HBsAg). In this
work, the effect of
pH,
ionic strength and
temperature on the native
state of the
HBsAg antigen were studied by a combination of biophysical
methods that included small angle
X-ray scattering,
synchrotron radiation circular dichroism,
fluorescence and
surface plasmon resonance spectroscopies, as well as in vivo and
in vitro potency assays. The native conformation, morphology,
radius of gyration, and antigenic properties of the
HBsAg antigen demonstrate high stability to
pH treatment, especially in the
pH range employed in all stages of
HBsAg vaccine production and storage. The
HBsAg protein presents thermal
melting point close to 56°C, reaching a more unfolded
state after crossing this point, but it only experiences loss of
vaccine potency and antigenic properties at 100°C. Interestingly, a 6-month storage period does not
affect vaccine stability, and the results are
similar when the
protein is kept under refrigerated conditions or at room
temperature (20°C). At frozen
temperatures, large aggregates (>200nm) are formed and possibly cause loss of
HBsAg content, but that does not
affect the in vivo assay. Furthermore,
HBsAg has a well-ordered
secondary structure content that is not affected when the
protein is formulated with
silica SBA-15, targeting the oral delivery of the
vaccine. The combined results from all the characterization
techniques employed in this study showed the high stability of the
antigen at different storage
temperature and extreme values of
pH. These findings are important for considering the delivery of
HBsAg to the
immune system via an oral
vaccine.