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. 2018 May 24;8:8078. doi: 10.1038/s41598-018-26281-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Immune responses following immunization with expression-optimized RT genes. BALB/c mice (n = 5 or 6) were immunized with two intradermal injections (29G needle) containing 20 µg of RTwt, RTwt-opt, RTwt-opt-in, RT1.14, RT1.14-opt, or RT1.14-opt-in encoding plasmids per mouse with subsequent electroporation by Dermavax (standard protocol) in two independent immunizations. At 21 days post-immunization mice were sacrificed, and sera and splenocytes were isolated for further immune tests. End-point average titers of anti-RT total IgG and IgG subtypes were detected using ELISA against recombinant RTwt and RT1.14 proteins with cut-offs set against the serum reactivity of control mice immunized with vector pVax 1. (a–c) ELISA with the identical RT protein variant received as a gene, i.e. recombinant RTwt protein for RTwt, RTwt-opt, and RTwt-opt-in immunized mice or RT1.14 for RT1.14, RT1.14-opt, and RT1.14-opt-in immunized mice. (a) ELISA with the homologous RT, i.e. recombinant RT1.14 protein for RTwt, RTwt-opt, and RTwt-opt-in or RTwt protein for RT1.14, RT1.14-opt, and RT1.14-opt-in immunized mice. (b) IgG2a/IgG1 ratio for antibody reactivity against RT variants matching RT used as DNA immunogen (c). In panels (a,b) the asterisk designates the difference between RTwt and RTwt-opt/RTwt-opt-in and between RT1.14 and RT1.14-opt/RT1.14-opt-in variants for all IgG subtypes. Murine splenocytes were stimulated in vitro with an RT-derived peptide representing the epitope of RT aa 528–543 (immunodominant T-cell epitope; Table 2) in the IFN-γ/IL-2 Fluorospot test (d–f). The average number of cells was registered as signal-forming units (sfu) per million splenocytes secreting IFN-γ (d) IL-2 (e) and IFN-γ/IL-2 (f) and the error bars represent the SD. *p < 0.05; **p < 0.01; tendency p-values are indicated on the graph. Statistical comparisons were performed using Kruskal-Wallis and Mann-Whitney tests.