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.

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines / Cavazzini, D.; Spagnoli, G.; Mariz, F. C.; Reggiani, F.; Maggi, S.; Franceschi, V.; Donofrio, G.; Muller, M.; Bolchi, A.; Ottonello, S.. - In: FRONTIERS IN IMMUNOLOGY. - ISSN 1664-3224. - 13:(2022), p. 958123. [10.3389/fimmu.2022.958123]

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines

Spagnoli G.;Reggiani F.;Maggi S.;Franceschi V.;Donofrio G.;Bolchi A.
;
Ottonello S.
2022

Abstract

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.
Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines / Cavazzini, D.; Spagnoli, G.; Mariz, F. C.; Reggiani, F.; Maggi, S.; Franceschi, V.; Donofrio, G.; Muller, M.; Bolchi, A.; Ottonello, S.. - In: FRONTIERS IN IMMUNOLOGY. - ISSN 1664-3224. - 13:(2022), p. 958123. [10.3389/fimmu.2022.958123]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2932159
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