Stability of Engineered Ferritin Nanovaccines Investigated by Combined Molecular Simulation and Experiments

Abstract

Human ferritin is regarded as an attractive and promising vaccine platform because of its uniform structure, good plasticity, and desirable thermal and chemical stabilities. Besides, it is biocompatible and presumed safe when used as a vaccine carrier. However, there is a lack of knowledge of how different antigen insertion sites on the ferritin nanocage impact the resulting protein stability and performance. To address this question, we selected Epstein–Barr nuclear antigen 1 as a model epitope and fused it at the DNA level with different insertion sites, namely, the N- and C-termini of ferritin, to engineer proteins E1F1 and F1E1, respectively. Protein properties including hydrophobicity and thermal, pH, and chemical stability were investigated both by molecular dynamics (MD) simulation and by experiments. Both methods demonstrate that the insertion site plays an important role in protein properties. The C-terminus insertion (F1E1) leads to a less hydrophobic surface and more tolerance to the external influence of high temperature, pH, and high concentration of chemical denaturants compared to N-terminus insertion (E1F1). Simulated protein hydrophobicity and thermal stability by MD were in high accordance with experimental results. Thus, MD simulation can be used as a valuable tool to engineer nanovaccine candidates, cutting down costs by reducing the experimental effort and accelerating vaccine design.

Immunogenicity and Vaccine Efficacy Boosted by Engineering Human Heavy Chain Ferritin and Chimeric Hepatitis B Virus Core Nanoparticles

“…The detailed information about plasmid construction for EBNA1−ferritin (E1F1) can be found in our previous research. 11 For EBNA1−HBc (E1H1), plasmid was built using the pET 30a vector. The restriction sites were Nde I and BamH I.…”

“…The restriction sites were Nde I and BamH I. 11 Information about protein sequence is shown in Table S1, Supporting Information. Escherichia coli BL21 (DE3) (Invitrogen, USA) was used to transform plasmids.…”

“…Bacterial solution was centrifuged to collect the bacteria pellet. The sonication followed the procedure 11 with lysis buffer (E1F1: 20 mM phosphate buffer (PB), 2 mM EDTA, pH 7.0; E1H1: 20 mM Tris-HCl, 3 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% Triton X-100, pH 8.0). Solubleexpressed protein was collected by centrifuging bacterial lysate at 13,751g for 30 min.…”

“…For the purification of E1F1, the supernatant of the cell lysate containing soluble expressed protein was diluted to protein concentration to 4 mg/mL with Mili-Q water. 11 The pH of the diluted supernatant was altered to 5.0 utilizing 1 M NaCl, NaAc−HAc (1 M, pH 5.0). Heating (60 °C, 10 min) was applied to precipitate protein impurities, followed by centrifugation (19,802g, 4 °C, 10 min) to remove protein impurities.…”

“…The procedure followed our previous research. 11 Briefly, the diluted samples (1 mg/mL) mixed with loading buffer 11 were disassembled by heating (100 °C, 10 min). Lanes were separated by electrophoresis (Bio-Rad, USA).…”