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. 2019 Oct 1;9:14085. doi: 10.1038/s41598-019-50365-z

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© The Author(s) 2019

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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FACS-based frequency analysis of antigen-specific cytokine producing T-cells after vaccination with HCV p7 protein or pepmix. Amino acid sequence of the HCV p7 protein or the six peptides comprising the corresponding pepmix, all based on HCV strain J4 (genotype 1b) (a). Vaccine antigens were formulated with the adjuvant CAF09. CB6F1 mice were vaccinated three times at 2-week intervals (b); control mice received CAF09 alone. PBMCs isolated from individual mice were re-stimulated with a p7 peptide pool corresponding to the amino acid sequence of the employed vaccine antigen and analyzed by multi-parameter FACS-analysis two weeks after the final vaccination. Representative FACS plots indicate the gating strategy on the IFN-γ (top panel), TNF-α (middle panel) and IL-2 (lower panel) producing population of CD44⁺ CD4⁺ T-cells (c) or CD44⁺ CD8⁺ T-cells (d). The bar charts show frequencies of p7-specific IFN-γ, TNF-α and IL-2 producing CD44⁺ CD4⁺ T-cells (e) and CD44⁺ CD8⁺ T-cells (f) out of the total CD4⁺ and CD8⁺ T-cell population, respectively. The data are shown as means and standard errors of the mean (SEM) (n = 4 to 8 mice in each vaccine group). *P < 0.05; **P < 0.01.