The biophysical nature and not only the size of protein aggregates determines the strength of the immune response against dengue ED3

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 aggregates can affect immunogenicity. We prepared amorphous oligomers in five distinct ways. 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. Immunization in JcL:ICR mice showed that both C5I and Ms significantly increased the anti-D3ED3 IgG titer. Ht, St, and FT were barely 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. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers. It also suggests that controlled oligomerization can provide a new, adjuvant-free method for increasing a proteins immunogenicity, yielding a potentially powerful platform for protein-based vaccines. SignificanceProtein aggregation is suspected to increase the immunogenicity of proteins. Here we show that the strength of the immune response depends not merely on the size of the oligomers/aggregates but also on their biophysical properties. Dengue virus 3 envelop protein domain 3 (D3ED3) was oligomerized/aggregated in five different ways. All five formulations contained oligomers with hydrodynamic radii between 30 and 55 nm. Two formulations, where D3 ED3 was natively folded, were not or poorly immunogenic. On the other hand, two others, where D3ED 3 was in a molten globule-like state, were strongly immunogenic. This result adds a new dimension to earlier studies where the strength of the immune response was associated solely with the presence and sizes of the oligomers.