%0 Journal Article
%A Cavazzini, Davide
%A Spagnoli, Gloria
%A Mariz, Filipe Colaco
%A Reggiani, Filippo
%A Maggi, Stefano
%A Franceschi, Valentina
%A Donofrio, Gaetano
%A Müller, Martin
%A Bolchi, Angelo
%A Ottonello, Simone
%T Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines.
%J Frontiers in immunology
%V 13
%@ 1664-3224
%C Lausanne
%I Frontiers Media
%M DKFZ-2022-02059
%P 958123
%D 2022
%X 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.
%K antigen carrier (Other)
%K epitope display (Other)
%K intracellular antigen delivery (Other)
%K peptide epitope (Other)
%K protein DNA interaction (Other)
%K protein scaffold engineering (Other)
%K protein scaffold vaccine (Other)
%K recombinant vaccine (Other)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:36032169
%2 pmc:PMC9405434
%R 10.3389/fimmu.2022.958123
%U https://inrepo02.dkfz.de/record/181524