Engineered Ferritin as a Nanoparticle Platform for Vaccine Delivery Via Molecular Dynamics Simulation and Experiment

Abstract

Researchers have spent decades developing safe and efficacious vaccine to protect and save lives. The current COVID-19 pandemic has sharply drawn attention to the need for innovative vaccine development. Traditional vaccines are based on live attenuated virus strains, or inactivated (killed) pathogens. Significant weaknesses with traditional vaccines include, low immunogenicity, and high risk of the virus replicating. Modern vaccines are developed to overcome these challenges. Recent studies have shown that epitope-based chimeric (EBC) vaccine is practically promising as one option because there is a broad selection of molecular sizes, highly repetitive structures to induce immune responses, and importantly, scalable and cost-effective production processes that are well developed. Human ferritin heavy chain (HFn), is structured by 24 identical subunits with each of molecular weight 21 kDa to form a spherical assembled structure with outer and inner diameters of 12 nm and 8 nm, respectively. HFn has a number of advantages as a vaccine carrier, including: it 1) has a robust thermal and chemical stability; 2) displays antigens in a well-organised manner to induce potent immune responses; and 3) is safe with good biocompatibility, biodegradability and low toxicity. Therefore, there is increasing interest in it as a protein nanocage to develop EBC. EBC vaccine consists of 3 main parts, protein nanocage (NPC), epitope and linker. Currently, approaches to develop vaccine rely significantly on extensive experiments that can result in high failure rates and costs. Additionally, there are limited studies focusing on molecular design of HFn (insertion sites, linker design and variant study) in order to enhance protein stability and boost vaccine immunogenicity. Significantly, a comparison of immunogenicity between HFn and other NPC, such as Hepatitis B Core (HBc) virus like particles (VLPs), is not yet reported. The overarching aim of this Thesis work was therefore to bridge these research gaps and apply molecular dynamics simulation (MDS) and judicious experiments to develop engineered HFn as a protein nanocage to develop novel EBC vaccine. The research focussed on 5 coordinated steps: 1) Molecular design of engineered ferritin inserted with epitopes at 2 different locations (N-terminus and C-terminus) and stability investigation; 2) Flexible linker length design to affect engineered ferritin stability against thermal- and chemical denaturants; 3) Engineered ferritin variant study via simulation and experiment; 4) Immunogenicity study of engineered ferritin inserted epitope at N- and C-terminus; and, 5) Immunogenicity study of HFn and HBc carrying the same epitope. In Step 1), model epitope Epstein-Barr Antigen 1 (EBNA1) was inserted at 2 locations, namely, N-terminus (E1F1) and C-terminus (F1E1). Protein surface hydrophobicity and thermal stability were predicted by MDS and validated by experiments. In Step 2), the effect of linker length on protein stability, short (3 residues) and long (15 residues) flexible linkers were inserted between the epitope and protein cage at N/C-terminus to form E1L15F1, F1L15E1, E1L3F1, and F1L3E1, respectively. Protein surface hydrophobicity and protein stability against thermal- and chemical denaturants were assessed experimentally. In Step 3), hot spots were predicted by MDS and variants (C1, C2, C3, C4 and C5) were designed to replace hot spots with other residues. C1 and C2 were built by replacing predicted hot spots with non-charged, or positive charged, residues. C3, C4 and C5 were constructed by replacing native hydrophobic residues with more hydrophobic or more hydrophilic residues. Molecular characterization, protein surface hydrophobicity and thermal stability by experiments were demonstrated. To investigate the effect of insertion site of HFn on vaccine immunogenicity, Step 4), characterization study was demonstrated to compare the structural difference between E1F1 and F1E1. IgG titer, proliferation index and memory T cells differentiation were performed to determine humoral and cell-mediated immune response induced by E1F1 and F1E1. In Step 5), a comparison of HFn NPC with HBc NPC, the immunogenicity of E1F1 (HFn NPC) was compared with E1H1 vaccine (HBc NPC) inserted with the same model epitope EBNA1. The key findings from this Thesis are: 1. A combined approach of MDS with experiment has been successfully demonstrated to design protein structure and predict protein stability. This approach can significantly reduce experimental cost and failure rate of designed vaccine. 2. Both MDS and experiment have shown that C-terminus insertion significantly boosts protein stability. This may potentially enhance vaccine efficacy over N-terminus insertion in vivo study. A more comprehensive vaccine efficacy study is required to further validate. However, this preliminary study is useful to provide guidance for design of NPC with multiple insertion sites. 3. Long flexible linker has less impact on protein stability against thermal and chemical denaturants when compared with short flexible linker that provides useful guidelines on designing linker length in EBC vaccine. 4. C-terminus variant study highlighted the importance of helix E on stabilising assembled protein conformational and thermal stability. This finding has increased understanding of HFn molecular structure in designing stable vaccine. 5. Compared with HBc-VLP, HFn is advantageous to significantly boost cell-mediated immune responses because of the stronger binding to T cell immunoglobulin. However, HFn induces weaker humoral and proliferative responses. Collectively these findings will significantly improve understanding in development of HFn as a vaccine carrier through processes of molecular design, vaccine stability and immunology. Research findings will therefore be of immediate practical benefit and interest to a wide range of researchers and manufacturers for innovative EBC vaccine development.