Development and Application of Synthetic Affinity Ligands for the Purification of Ferritin-Based Influenza Antigens

Abstract

A recently developed novel recombinant influenza antigen vaccine has shown great success in preclinical studies in ferrets and mice. It provides broader protection, and is efficient to manufacture compared to the conventional trivalent influenza vaccines (TIV). Each strain of the recombinant antigen has a constant self-assembled bacterial ferritin core which, if used as a target for affinity chromatography, could lead to a universal purification method. Ferritin in silico models were used to explore potential target binding sites against ligands synthesized by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesized in solution, characterized by H,C, and 2D NMR spectroscopy, and subsequently immobilized on the PEG-functionalized beads. Ligands SJ047 and SJ055 displayed apparent K values of 2.04 × 10 M and 1.91 × 10 M, respectively, against the ferritin. SJ047 and SJ055-functionalized resins were able to purify hemagglutinin (New Caledonia)-ferritin expressed in a crude Human Embryonic Kidney (HEK) cell supernatant in a single step to a purity of 85 ± 0.5% (97 ± 1% yield) and 87.5 ± 0.5% (95.5 ± 1.5% yield), respectively. Additionally, SJ047 and SJ055-functionalized resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, hemagglutinin (New Caledonia)-ferritin, hemagglutinin (California)-ferritin, and hemagglutinin (Singapore)-ferritin were purified, thereby offering an ideal alternate platform for affinity chromatography. Following elution from the affinity adsorbents, absorbance at 350 nm showed that there was no aggregation of the recombinant antigens and dynamic light scattering studies further confirmed the structural integrity of the recombinant antigen. The use of Ugi ligands coupled to a PEG-spacer arm to target the ferritin core of the strain is entirely novel and provides an efficient purification of these recombinant antigens. This approach represents a potentially universal method to purify any ferritin-based vaccine.

Image analysis techniques to map pyramids, pyramid structure, glomerular distribution, and pathology in the intact human kidney from 3-D MRI

“…Ferritin-based contrast agents can be made recombinantly (34) using the human ferritin sequence. Furthermore, ferritin protein has been used as a vehicle in vaccinations such as influenza, human papillomavirus, and more recently COVID-19 (58)(59)(60). We have shown that CF can also be converted to a glomerulus-specific radiolabeled contrast agent for positron emission tomography, detectable in trace doses (<1/1,000th of that used in CFE-MRI) (32).…”

Functionalizing Ferritin Nanoparticles for Vaccine Development

“…Instead of using the binding capacity of the antigens, Jacob et al [ 98 ] developed a novel method to purify ferritin-based vaccines by using the ferritin core as a target for affinity chromatography. Multicomponent Ugi ligands, with affinity to hydrophobicity sites on ferritin, were coupled to the resin; due to the presence of HA trimers on the surface of ferritin, a PEG-spacer arm was added to enable accessibility to the ferritin.…”

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft

“…The appropriate surface modification enables nanoparticles to reside stably in the blood circulatory system [ 21 ]. Characterized by a large surface area and a hollow spherical structure, Ferritin (Ft) was reported to form nanoparticles of 24 identical polypeptides by self-assembling with a variety of expressed fusion proteins, which promoted antigen recognition and uptake by antigen-presenting cells, thereby inducing a strong immune response [ 22 , 23 ]. Ferritin is widely applied in vaccine development.…”