Immunogenicity study of engineered ferritins with C- and N-terminus insertion of Epstein-Barr nuclear antigen 1 epitope

Human ferritin heavy chain, an example of a protein nanoparticle, has recently been used as a vaccine delivery platform. Human ferritin has advantages of uniform architecture, robust thermal and chemical stabilities, and good biocompatibility and biodegradation. There is however a lack of understanding about the relationship between insertion sites in ferritin (N-terminus and C-terminus) and the corresponding humoral and cell-mediated immune responses. To bridge this gap, we utilized an Epstein-Barr Nuclear Antigen 1 (EBNA1) epitope as a model to produce engineered ferritin-based vaccines E1F1 (N-terminus insertion) and F1E1 (C-terminus insertion) for the prevention of Epstein-Barr virus (EBV) infections. X-ray crystallography confirmed the relative positions of the N-terminus insertion and C-terminus insertion. For N-terminus insertion, the epitopes were located on the exterior surface of ferritin, while for C-terminus insertion, the epitopes were inside the ferritin cage. Based on the results of antigen-specific antibody titers from in-vivo tests, we found that there was no obvious difference on humoral immune responses between N-terminus and C-terminus insertion. We also evaluated splenocyte proliferation and memory lymphocyte T cell differentiation. Both results suggested C-terminus insertion produced a stronger proliferative response and cell-mediated immune response than N-terminus insertion. C-terminus insertion of EBNA1 epitope was also processed more efficiently by dendritic cells (DCs) than N-terminus insertion. This provides new insight into the relationship between the insertion site and immunogenicity of ferritin nanoparticle vaccines.