Simplified Purification of Glycoprotein-Modified Ferritin Nanoparticles for Vaccine Development

Abstract

Ferritin-based, self-assembling protein nanoparticle vaccines are being developed against a range of viral pathogens, including SARS-CoV-2, influenza, HIV-1, and Epstein-Barr virus. However, purification of these nanoparticles is often laborious and requires customization for each potential nanoparticle vaccine. We propose that the simple insertion of a polyhistidine tag into exposed flexible loops on the ferritin surface (His-Fer) can mitigate the need for complex purifications and enable facile metal-chelate-based purification, thereby allowing for optimization of early stage vaccine candidates. Using sequence homology and computational modeling, we identify four sites that can accommodate insertion of a polyhistidine tag and demonstrate purification of both hemagglutinin-modified and SARS-CoV-2 spike-modified ferritins, highlighting the generality of the approach. A site at the 4-fold axis of symmetry enables optimal purification of both protein nanoparticles. We demonstrate improved purification through modulating the polyhistidine length and optimizing both the metal cation and the resin type. Finally, we show that purified His-Fer proteins remain multimeric and elicit robust immune responses similar to those of their wild-type counterparts. Collectively, this work provides a simplified purification scheme for ferritin-based vaccines.

A ferritin-based COVID-19 nanoparticle vaccine that elicits robust, durable, broad-spectrum neutralizing antisera in non-human primates

“…Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-housemade primary antibody, CR3022 as previously described 14,70 (approximate concentration 0.8-1.3 mg/mL), was added at a 1:10,000 dilution in PBST. Blots were washed with PBST, and secondary rabbit antihuman IgG H&L HRP (abcam ab6759) was added at 1:10,000 in PBST.…”

Ferritin nanocages as efficient nanocarriers and promising platforms for COVID-19 and other vaccines development

“…Following precipitation and clarification steps, bacterial derived ferritin may be further purified through size-exclusion chromatography (SEC), differential centrifugation and/or ion-exchange chromatography (IEC) [ 75 ]. The precipitation steps may be skipped entirely to preserve the integrity of antigen/epitope whereby ferritin nanoparticles may be separated by (i) affinity chromatography using an inserted His-tag on the protein [ 76 ] or (ii) IEC followed by SEC [ 75 ]. On the other hand, purification of ferritin from animal cell cultures is rather straightforward, requiring simple centrifugation or filtration followed by further purification through chromatographic approaches [ 75 ].…”

A conserved N protein nano‐vaccine of COVID‐19 exerts potent and cross‐reactive humoral and cellular immune responses in mice

“…To promote the immunogenicity of the N protein, we fused it at the N-terminal of H. pylori nonheme Ferritin to display the N on the Ferritin nanoparticles (Figures 1A and 1B), fused protein of target protein and Ferritin have the ability to self-assemble into 24-mer and have been applied to HIV, influenza, and HPV vaccine research. [22][23][24] The N and N-Ferritin were expressed in E. coli and purified by using Ni-TED 6FF. The purity levels of the Ferritin, N protein, and N-Ferritin were verified by using Coomassie blue staining and western blot analysis (Figure 1C,D).…”