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. 2019 May 15;10:1015. doi: 10.3389/fimmu.2019.01015

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Copyright © 2019 Mohsen, Vogel, Riether, Muller, Salatino, Ternette, Gomes, Cabral-Miranda, El-Turabi, Ruedl, Kundig, Dermime, Knuth, Speiser and Bachmann.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Bio-orthogonal Cu-free click chemistry; an efficient method for coupling antigens to VLPs to enhance their immunogenicity. (A) A sketch illustrating the coupling method using Bio-orthogonal Cu-free click chemistry (dibenzocyclooctyne NHS ester DBCO). Briefly, NHS ester reacts with Lys residues on VLPs and incorporates a cyclooctyne moiety which reacts with the azide labeled molecule forming a stable triazole linkage. (B) SDS-PAGE stained with coomassie blue showing the coupling efficiency of p33 to Qβ(CpGs)-VLP using SMPH cross-linker or DBCO cross-linker. Lane 1 protein marker, lane 2 Qβ-VLP monomer, lane 3 Qβ(CpGs)-VLP derivatized with SMPH cross-linker, lane 4 Qβ(CpGs)-p33 vaccine using SMPH cross-linker, lane 5 Qβ(CpGs)-VLP derivatized with DBCO cross-linker, lane 6 Qβ(CpGs)-p33 vaccine using DBCO cross-linker. Each extra band in lanes 4 and 6 indicates a peptide binds to a Qβ monomer. (C) Densitometric analysis of SDS-PAGE lanes 1, 4, and 6. (1) uncoupled Qβ-VLP, total 180 monomers, (2) Qβ(CpGs)-p33 vaccine using SMPH cross-linker, and (3) Qβ(CpGs)-p33 vaccine using DBCO cross-linker. Notice the percentage of the coupled peptides to Qβ-VLP monomers, total 180 monomers. (D) Percentage of CD8⁺ Tetramer⁺ cells (means ± SEM) in the spleen of vaccinated groups: group 1) Qβ(CpGs)-VLPs, group 2) Qβ(CpGs)-p33 vaccine using SMPH cross-linker, and group 3) Qβ(CpGs)-p33 vaccine using DBCO cross-linker. (E) Percentage of CD8⁺ IFN-γ⁺ secreting cells (means ± SEM) in the spleen of vaccinated groups. (F) CFSE in vivo lytic activity in the spleen of vaccinated groups, using the formula 100X(1–CFSE^Hi pulsed/CSFE^Low un-pulsed). Statistical analysis by Students t-test. (G) Representative FACS plots showing the percentage of CD8⁺ Tetramer⁺ cells in the spleen of vaccinated groups. (H) Representative FACS histogram of CFSE in vivo lytic activity in the spleen of vaccinated groups. (n = 3) mice per group, one representative of 3 similar experiments is shown. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001.