Comparison of different epitope prediction methods for in silico spacer design based on the polypeptide proposed by Levy et al. Spacer sequences were constructed with SYFPEITHI, BIMAS, and SMM. Cleavage prediction was performed with PCM, classifying a site as cleaved if its score was greater than zero. The epitope thresholds used for neo-epitope detection were SYFPETHI-score ≥ 20, BIMAS ≥ 100 T 1/2, and SMM ≤ 500 nM. Red bars represent predicted epitopes and the intensity indicates overlapping epitopes at that position. The blue rectangles represent predicted C-terminal cleavage sites. Spacer sequences are marked in red. A tick indicates the start position of a predicted nine-mer epitope. Although, the different prediction methods yielded different spacer sequences, the overall result remained the same. The in silico designed spacers were superior in terms of recovered epitopes and neo-epitope formation. (PDF 1198 kb)