Introduction
The Manual for EpiQuest-T program explains in detail how to use the program and change the settings, if required. Here we show the analysis of several sequences included into Demo Sequence for the program illustrating the precision of the program and problems that may occur during interpretation of the results. We present here the example of prediction HLA-A2-restricted human CTL epitopes, more examples and discussion of other cases will be published regularly in our blog.
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The results are easy to read and interpret for molecules that contain one main CTL epitope (in this case, 2 overlapping, both located in 1-12 sequence of the molecule (marked by the red frame in the histogram)). As can be seen, the area containing the epitopes was successfully discovered by the program, and no additional falsely positive areas were detected (predicted).
HLA-A2: CTL-recognized antigen on melanoma (CAMEL) [Homo sapiens]
The CTL epitopes of CAMEL were described in: Aarnoudse, C.A., van den Doel, P.B., Heemskerk, B., and Schrier, P.I. (1999). Interleukin-2-induced, melanoma-specific T cells recognize CAMEL, an unexpected translation product of LAGE-1. Int J Cancer 82, 442–448.
HLA-A2: Melanoma antigen recognized by T-cells 1 [Homo sapiens]
Similar result is obtained for MART-1 antigen containing only one immunodominant HLA-A2 restricted CTL epitope: EpiQuest-T predicts the area corresponding to exact location of the epitope, only slightly wider.
The experimental data of CTL epitopes of MART1 are from: Borbulevych, O.Y., Insaidoo, F.K., Baxter, T.K., Powell, D.J., Johnson, L.A., Restifo, N.P., and Baker, B.M. (2007). Structures of MART-126/27-35 Peptide/HLA-A2 complexes reveal a remarkable disconnect between antigen structural homology and T cell recognition. J Mol Biol 372, 1123–1136.
HLA-A2: Transcriptional activator Tax [Human T-lymphotropic virus 1]
The sequences where multiple epitopes but of low strength are present are more difficult to analyse. The Analysis or Tax molecule in the Default settings show only one area fully overlapping with the actual CTL epitopes (the C-terminal one), the other two areas only partially overlap with the actual CTL epitopes (click on the image to zoom the view). The bar below the histogram only shows positive peptides of length of 8 aa and above, therefore areas of low AGI will not be reported at Default settings, however, the presence of some peaks suggests that there may be CTL epitopes of lower strength.
In such cases we recommend to increase the sensitivity of the assay by lowering the Threshold of detection (T to -1). As a result, several larger areas of potential epitopes are discovered (which include all experimentally discovered epitopes of Tax.
The experimental data on CTL epitopes of Tax and their activity are from: Sundaram, R., Sun, Y., Walker, C.M., Lemonnier, F.A., Jacobson, S., and Kaumaya, P.T.P. (2003). A novel multivalent human CTL peptide construct elicits robust cellular immune responses in HLA-A*0201 transgenic mice: implications for HTLV-1 vaccine design. Vaccine 21, 2767–2781.
HLA-A2: Insulin, Precursor [Homo sapiens]
Here we illustrate how changing the settings for analysis (the Frame and the Threshold) affect the results.
In general we recommend using Frame (F)=9 and Threshold (T)=0, these are the default settings. The top image shows the analysis of Insulin sequence at the default settings, the main (strongest) CTL epitope for HLA-A2 is indicated by the red frame (we do not discuss here the weaker epitopes that are present in the insulin molecule and are detected by the program). You can see that the whole area 1-16 is detected by the program without discriminating the epitope 6-14 from other weaker epitopes. Decreasing the Frame size usually allows to better discriminate the stronger epitope, as it lowers the influence of the context on the position of epitope. Using Frame=6 we can see more pronounced peak corresponding to epitope 6-14 (the middle image). If you increase the threshold (T=1, lower picture) the separation of the peak is even more clear.
The Insulin molecule contains a dominant epitope 6-14 and a weaker one 2-10. The souce of data: Toma, A., Laïka, T., Haddouk, S., Luce, S., Briand, J.-P., Camoin, L., Connan, F., Lambert, M., Caillat-Zucman, S., Carel, J.-C., et al. (2009). Recognition of human proinsulin leader sequence by class I-restricted T-cells in HLA-A*0201 transgenic mice and in human type 1 diabetes. Diabetes 58, 394–402.
HLA-A2: Nucleocapsid protein [SARS coronavirus]
Here we show the results of analysis of Nucleocapsid protein for SARS. It contains one strong epitope (red frame), which is correctly predicted by EpiQuest-T using the Default settings. However, the area of the dominant epitope predicted by the program is shifted from to the left from the position of the actual epitope. We do observe this sometimes when the epitope area is followed by a highly negative sequence. Therefore we suggest also testing the sequence at higher sensitivity settings ( T=-1 or even T=-2) to see the wider region surrounding the sequence detected at the original settings. In this case the entire epitope sequence will likely to be included (for initial functional tests, we suggest to take a peptide that is a bit longer then peak observed at the default settings is low.)
SARS CTL epitope data are from: Cheung, Y.K., Cheng, S.C.S., Sin, F.W.Y., Chan, K.T., and Xie, Y. (2008). Investigation of immunogenic T-cell epitopes in SARS virus nucleocapsid protein and their role in the prevention and treatment of SARS infection. Hong Kong Med J 14 Suppl 4, 27–30.
In all shown cases the tabular results correspond to the bars shown in the histogram. These bars unite overlapping peaks and show only sequences of 8aa and longer (the default settings, may be changed), as this is a minimal size of CTL epitope. The tabular results show also the AGI for predicted regions. In the example below we show the tabular results of the analysis of Glycoprotein of HPV-8 which contains one immunodominant epitope at position 736-745. In tabular result you can see that the sequence 730-747 is indicated as the most probable to contain a dominant epitope, and its AGI is twice as high as for any other detected sequences.
HLA-A2: Glycoprotein B-like [Human herpesvirus 8]
Source of epitope data: Lepone, L., Rappocciolo, G., Knowlton, E., Jais, M., Piazza, P., Jenkins, F.J., and Rinaldo, C.R. (2010). Monofunctional and polyfunctional CD8+ T cell responses to human herpesvirus 8 lytic and latency proteins. Clin Vaccine Immunol 17, 1507–1516.
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