Biophysical and biochemical nature of amorphous protein oligomers determines the strength of immune response and the generation of T-cell memory

Here, we used domain 3 of dengue virus serotype 3 envelope protein (D3ED3), a natively folded globular low-immunogenicity protein, to ask whether the biophysical nature of amorphous oligomers can affect immunogenicity. We prepared nearly identical 30 ~ 50 nm-sized amorphous oligomers in five distinct ways and looked at any correlation between their biophysical properties and immunogenicity. One oligomer type was produced using our SCP tag (solubility controlling peptide) made of 5 isoleucines (C5I). The others were prepared by miss-shuffling the SS bonds (Ms), heating (Ht), stirring (St) and freeze-thaw (FT). Dynamic light scattering showed that all five formulations contained oligomers of approximately identical sizes with hydrodynamic radii (Rh) between 30 and 55 nm. Circular dichroism (cd) indicated that the secondary structure content of oligomers formed by stirring and freeze-thaw was essentially identical to that of the native monomeric D3ED3. The secondary structure content of the Ms showed moderate changes, whereas the C5I and heat-induced (Ht) oligomers exhibited a significant change. The Ms contained D3ED3 with intermolecular SS bonds as assessed by nonreducing size exclusion chromatography (SEC). Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titre. Ht, St and FT were only mildly immunogenic, similar to the monomeric D3ED3. Cell surface CD marker analysis by flow cytometry confirmed that immunization with Ms generated a strong central and effector T-cell memory. Our observations indeed suggest that controlled oligomerization can provide a new, adjuvant-free method for increasing a protein's immunogenicity, yielding a potentially powerful platform for protein-based (subunit) vaccines.