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In this paper, we describe the methodologies behind three different aspects of the NetMHC family for prediction of MHC class I binding, mainly to HLAs. We have updated the prediction servers, NetMHC-3.2, NetMHCpan-2.2, and a new consensus method, NetMHCcons, which, in their previous versions, have been evaluated to be among the very best performing MHC:peptide binding predictors available. Here we describe the background for these methods, and the rationale behind the different optimization steps implemented in the methods. We go through the practical use of the methods, which are publicly available in the form of relatively fast and simple web interfaces. Furthermore, we will review results obtained in actual epitope discovery projects where previous implementations of the described methods have been used in the initial selection of potential epitopes. Selected potential epitopes were all evaluated experimentally using ex vivo assays., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Motivation: Immunological bioinformatics methods are applicable to a broad range of scientific areas. The specifics of how and where they might be implemented have recently been reviewed in the literature. However, the background and concerns for selecting between the different available methods have so far not been adequately covered., Summary: Before using predictions systems, it is necessary to not only understand how the methods are constructed but also their strength and limitations. The prediction systems in humoral epitope discovery are still in their infancy, but have reached a reasonable level of predictive strength. In cellular immunology, MHC class I binding predictions are now very strong and cover most of the known HLA specificities. These systems work well for epitope discovery, and predictions of the MHC class I pathway have been further improved by integration with state-of-the-art prediction tools for proteasomal cleavage and TAP binding. By comparison, class II MHC binding predictions have not developed to a comparable accuracy level, but new tools have emerged that deliver significantly improved predictions not only in terms of accuracy, but also in MHC specificity coverage. Simulation systems and mathematical modeling are also now beginning to reach a level where these methods will be able to answer more complex immunological questions.

Background: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein. It does so by integrating predictions of proteasomal cleavage, TAP transport efficiency, and MHC class I affinity. At least four other methods have been developed recently that likewise attempt to predict CTL epitopes: EpiJen, MAPPP, MHC-pathway, and WAPP. In order to compare the performance of prediction methods, objective benchmarks and standardized performance measures are needed. Here, we develop such large-scale benchmark and corresponding performance measures and report the performance of an updated version 1.2 of NetCTL in comparison with the four other methods., Results: We define a number of performance measures that can handle the different types of output data from the five methods. We use two evaluation datasets consisting of known HIV CTL epitopes and their source proteins. The source proteins are split into all possible 9 mers and except for annotated epitopes; all other 9 mers are considered non-epitopes. In the RANK measure, we compare two methods at a time and count how often each of the methods rank the epitope highest. In another measure, we find the specificity of the methods at three predefined sensitivity values. Lastly, for each method, we calculate the percentage of known epitopes that rank within the 5% peptides with the highest predicted score., Conclusion: NetCTL-1.2 is demonstrated to have a higher predictive performance than EpiJen, MAPPP, MHC-pathway, and WAPP on all performance measures. The higher performance of NetCTL-1.2 as compared to EpiJen and MHC-pathway is, however, not statistically significant on all measures. In the large-scale benchmark calculation consisting of 216 known HIV epitopes covering all 12 recognized HLA supertypes, the NetCTL-1.2 method was shown to have a sensitivity among the 5% top-scoring peptides above 0.72. On this dataset, the best of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL. All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php.

High-performing MHC class I binding predictions have been available for more than a decade; however, their value in terms of actual epitope finding has only now been estimated in a large-scale investigation undertaken by the group of Sette. This work underlines the importance of bioinformatics as a resource-saving tool in the field of epitope discovery. In addition, the data can be used to benchmark the performance of other new or existing CTL epitope-prediction tools.