Additional file 1 of A multiple T cell epitope comprising DNA vaccine boosts the protective efficacy of Bacillus Calmette–Guérin (BCG) against Mycobacterium tuberculosis
Creators: name: Sudeep Kumar Maurya
name: Aqdas, Mohammad
name: Deepjyoti Kumar Das
name: Sanpreet Singh
name: Nadeem, Sajid
name: Kaur, Gurpreet
name: Agrewala, Javed Naim
Publisher: name: figshare
Year: 2020
Subjects: subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine

description: Additional file 1: Supplementary Figure 1. C6 gene construction. (a) All the peptide sequences were analyzed by PROSPER software to check their sensitivity to proteases. Different colors indicate different proteases. ‘Red colour’ designates the sequence of Rv0476, which was found to be most sensitive to protease degradation. (b) The complete gene sequence of C6 (963bp) is shown that was used for the synthesis of the DNA vaccine. Supplementary Figure 2. C6 was cloned successfully into the plasmid. (a) The C6 gene was cloned into pcDNA3.1- and transformed into E. coli. The agarose gel electrophoresis confirms the presence of the C6 gene with the additional size of the restriction site and extra-base pairs used for restriction digestion. C6: positive control, lane 1–8: different E. coli clones. The vector map of pcDNA3.1-C6 with the site was used for cloning. (b) The C6 gene was cloned into a pEYFP-N1 plasmid and transformed into E. coli. The agarose gel electrophoresis confirms positive E. coli colonies with the C6 gene. Lane 1–10 indicates the different E. coli colonies and C indicates the positive control of the C6 gene. The vector map of pEYFP-C6 was used for cloning and as an open reading frame. (c) The C6YFP gene was cloned into pcDNA3.1- plasmid and transformed into E. coli. The agarose gel electrophoresis confirms positive E. coli colonies with the C6 gene. Lane 1–6 indicate the different E. coli colonies. pEYFP-C6 vector was used as a positive control. The vector map of pcDNA3.1-C6YFP with the site was used for cloning and as an open reading frame. Supplementary Figure 3. C6-YFP expression in CHO cells. (a) The C6-YFP transfected CHO cells were analyzed by flow cytometry for their fluorescence intensity (YFP) and represented as histogram plots. Data in the inset represent the mean fluorescence intensity (MFI). (b) The bar diagram represents the integrated mean fluorescence intensity (iMFI) of the transfected cells, further, confirm the presence of C6. Data expressed as mean ± SEM are representative of 2 independent experiments. * p ≤ 0.05. Supplementary Figure 4. Co-immunization of BCG with C6 augments memory T cell response. Lymphocytes from the lungs were isolated from the BCG + C6 injected and control animals. Lymphocytes were in vitro stimulated with PPD and peptides. (a, c) Contour plots show the percent population of CD62LhiCD44hi central memory CD4 T cells and CD8 T cells, stimulated with (a) PPD and (c) peptides and represented as (b, d) bar diagrams. Data are from two independent experiments and represented as mean ± SEM. Supplementary Figure 5. Prime boosting with BCG and C6 enhances the activation of DCs and macrophages. Lymphocytes were isolated from spleen and LNs from the BCG + C6 immunized and control animals. Cells were in vitro stimulated with LPS (1 μg/ml) for 24 h. (a) Gating strategy used for DCs and macrophage cells. (b, c) histogram and their respective (d, e) bar diagram signify the percent population of CD86hi, CD40hi, MHC-IIhi, and CD80hi expressing (b) DCs and (c) macrophages. Data (means±SEM) represented as percent positive cells are of two independent experiments. *p ≤ 0.05, **p ≤ 0.005, ***p ≤ 0.0005, ****p ≤ 0.0001.