Journal
FRONTIERS IN IMMUNOLOGY
Volume 13, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fimmu.2022.958123
Keywords
antigen carrier; recombinant vaccine; peptide epitope; intracellular antigen delivery; epitope display; protein scaffold engineering; protein scaffold vaccine; protein DNA interaction
Categories
Funding
- Interuniversity Consortium for Biotechnology (CIB)
- Baden-Wuerttemberg Stiftung
- Italian Ministry for Education, University and Research (MIUR)
- University of Parma
- [WSF-030]
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In this study, a positively supercharged protein was constructed and characterized as a promising carrier for intracellular antigen delivery. The protein showed different intracellular localization and cell-penetrating ability. Fusion of peptide epitopes to the protein enhanced its immunogenicity and induced a faster immune response.
Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.
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