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CTL Epitope Mapping

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Improved Strategies for Epitope Mapping
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Libraries of truncated peptides are powerful tools for identifying the peptides recognized by T-cells. Cytotoxic T lymphocytes (CTL) recognize an epitope comprised of a self portion (Class I MHC) and a foreign portion (a short peptide). It is known that the foreign peptide part of CTL epitopes consists of peptides using between 8 and 11 residues in length 1. See Figure 2 for a diagramatic illustration of the binding of a 9-mer to a Class I MHC molecule.

Previous epitope mapping strategies for CTL using synthetic peptides were imprecise and relied on a breakdown process acting on the peptides, either in the culture medium or in the cells, to generate the active peptides from longer sequences2,3. Another approach, use of predictive methods, is effective at locating substantial numbers of CTL epitopes when the binding motifs of all the Class I MHC isotypes and allotypes are known, but even then there can be epitopes which do not conform exactly to consensus motifs, or which vary in length. To do this with conventional peptide synthesizers is costly and impractical because of the large number of peptides and the large amount of testing required.


Improved Strategies for Epitope Mapping

PepSets™ Peptide Libraries are powerful tools for identifying the peptides recognized by T-cells. Mapping cytotoxic T-cell (CTL or Tc) epitopes of a whole protein can be difficult because of the large number of possible active sequences, and the unknown length requirements for MHC binding and bioactivity (whether 8, 9, 10 or 11 residue peptides).

This article presents the use of a novel technology for the synthesis and testing of CTL epitopes using PepSets Peptide Libraries in which each nominal 11-mer peptide is made up of an equimolar mixture of the four C-terminal peptides comprising the 8-mer to 11-mer peptides. Because the library contains all the possible active CTL epitopes, it is more effective than longer peptides for mapping cytotoxic T-cells and gives a more precise answer from the initial CTL epitope screening experiments.

The Truncated PepSets Peptide Library PepSets-Tc overcomes previous limitations by allowing the researcher to test all possible peptides homologous with the protein of interest, with lengths between 8 and 11 residues.



Mimotopes’ proprietary technology has been applied to the PepSets Peptide Library and devised a method of synthesizing, in one tube, an equimolar mixture of peptides of any series of lengths, provided they have a shared C-terminal sequence.

By making the required number of peptide tubes to span the sequence (around the same as the number of residues in the protein), all possible 8, 9, 10 and 11-mer peptides of the protein are present in similar quantity. These peptides can be further pooled for assay, knowing that because all potential epitopes are present, all epitopes will be efficiently detected, even at very low added doses of peptide or in the presence of other peptides. See Figure 1 for a diagrammatic representation of the composition of each tube for such a CTL mapping set.

To compare the effectiveness of a Truncated PepSet against the effectiveness of a conventional set of peptides, the overlapping peptides corresponding to the F (fusion) protein of Respiratory Syncytial virus (RSV), a protein of 574 AA residues, were made in two formats. The first format was a set of overlapping peptides in which each tube contained a single 14-mer peptide. The second format was a set of overlapping truncated peptides in which each tube contained the 8-mer, 9-mer and 10-mer sequences. These two sets were compared for effectiveness at detection of cytotoxic T-cells in ELISPOT using human peripheral blood mononuclear cell preparations.



The peptide mixtures can be used directly in gamma interferon ELISPOT, an assay for CTL activation. In this early study, the Truncated PepSet was a set of 565 10-mers (overlap of 9 residues) rather than 11-mers with an overlap of 10 residues. Each nominal 10-mer contained the 9-mer and 8-mer, and it was compared with a set of 188 non-truncated 14-mer peptides with an overlap of 11 residues.

The sixteen subjects were healthy volunteers known to be immune to RSV. Table 1 shows the comparison of the frequency of positive ELISPOT data summed by peptide and donor, i.e. each instance of a positive ELISPOT reading with a donor and a peptide preparation was scored as 1, and summed to give the totals shown. It can be seen that, as expected, there were many more positive peptide/subject combinations from the truncated peptides than from the single 14-mers.

This is expected because two successive 10-mer peptides can easily span a single CTL epitope, for example if it were a 9-mer, then potentially, two contiguous overlapping 10-mers could be stimulatory. More significant however is the observation that the truncated 10-mers revealed 35 unique stimulatory sites along the F protein sequence, many more than the 14-mers. This is despite the possibility that at least some of the positive data from the 14-mers may have been due to Th (T helper) cells, whereas the ELISPOT with the truncated 10-mers almost certainly is due to Tc because 10-mers have been generally found to be too short to act as effective Th epitopes.

Subsequent work 4 has shown that a strongly recognized peptide from this protein, sequence RELPRFMNYT, is a frequently recognized CTL epitope seen in the context of the HLA A*0101 MHC Class I allotype. Significantly, this epitope was not detected in initial screens with the 14-mer peptides spanning this sequence. The higher frequency of detection of ELISPOT positive peptides with PepSet-Tc peptides is a clear indication that the truncated 10-mers are more efficient at priming antigen-presenting cells with externally added peptide than the 14-mers, and thus are a more effective reagent for discovering biologically signficant Tc epitopes.

Figure 2 shows the analytical LC profile of the peptides in one tube of a truncated peptide containing the 8-mer, 9-mer, 10-mer and 11-mer of the sequence of interest. It is seen that there are four major peaks, corresponding to the full length sequence and the three truncated versions of the full length peptide.



Further pooling of truncated peptide mixtures can reduce the amount of screening work needed 5. When stimulatory pools are found, the active peptide mixture can be deduced by testing the individual samples which had been used to prepare the stimulatory pool. It has been shown that mixing of CTL epitope peptides does not cause masking of the active sequences 5. Strategies for pooling and the advantages of doing so have been explained in some detail 6. In another recent study with truncated peptides 7, it was confirmed that there is a single CTL epitope in the E7 oncoprotein of human papillomavirus type 16. This study also included the use of a combination of phosphorylated and non-phosphorylated amino acids, necessary because the protein is known to be at least partly phosphorylated in vivo.



Sets of short, deliberately truncated peptides are much more effective for mapping Tc epitopes than longer single peptides spanning the same sequence. This is expected on theoretical grounds and was confirmed in an initial screening study.



  1. Rammensee, H.G., Friede, T. and Stevanovic, S. (1995) MHC ligands and peptide motifs: first listing. Immunogenetics 41, 178.
  2. Luckey, C.J., King, G.M., Marto, J.A., Venketeswaran, S., Maier, B.F., Crotzer, V.L., Colella, T.A., Shabanowitz, J., Hunt, D.F. and Engelhard, V.H. (1998) Proteasomes can either generate or destroy MHC class I epitopes: evidence for nonproteasomal epitope generation in the cytosol. J. Immunol. 161, 112.
  3. Maecker, H.T., Dunn, H.S., Suni, M.A., Khatamzas, E., Pitcher, C.J., Bunde, T., Persaud, N., Trigona, W., Fu, T., Sinclair, E., Bredt, B.M., McCune, J.M., Maino, V.C., Kern, F. and Picker, L.J. (2001) Use of overlapping peptide mixtures as antigens for cytokine flow cytometry. J. Immunol. Methods 255, 27.
  4. Rock, M.T. and Crowe, J.E. (2003) Identification of a novel human leucocyte antigen-A*01-restricted cytotoxic T-lymphocyte epitope in the respiratory syncytial virus fusion protein. Immunology 108, 474.
  5. Sun Y., Iglesias E., Samri A., Kamkamidze G., Decoville T., Carcelain G. and Autran B. (2003) A systematic comparison of methods to measure HIV-1 specific CD8 T cells. J. Immunol. Methods 272, 23.
  6. Roederer M. and Koup, R.A. (2003) Optimized determination of T cell epitope responses. J. Immunol. Methods 274, 221.
  7. Khammanivong, V., Liu, X.S., Liu, W.J., Rodda, S.J., Leggatt, G.R., Tindle, R.W., Frazer, I.H. and Fernando, G.J.P. (2003) Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16. Immunol. Cell Biol. 81, 1.
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