where Xi means the times that the given amino acid X appears at the position i of the alignment and N is the number of sequences in the alignment. All frequencies are compared to a threshold value, which is 25% by default. If a frequency is more than the threshold, the corresponding residue is considered as a motif residue at that position. If the sum frequency of similar residue at the same position is above the threshold, the similar residues are also regarded as motif residues. In MIMOX, there are five similar residue groups (L, I, V; T, S; E, D; Q, N; K, R; F, W); other residues are considered unique. This classification scheme is the same as that used by Mapitope[16,17]. If no motif residue is found at a given position, then X is used to stand for any amino acid residue. Motif residues at all positions of the alignment are then displayed in a table. Thus a consensus sequence is suggested by the program. The script also creates a 3D bar figure based on the statistical analysis above, where the X axis represents the 20 amino acid types and gap type, Y axis is the frequency and Z axis stands for the position of the aligned sequences (Shown in Figure ​Figure22).

The first case is taken from FINDMAP[13]. In 1999, Jesaitis and co-workers used an anti-actin polyclonal antibody to select a phage displayed random peptide library; VPHPTWMR was one of the consensus sequences they derived from the selected mimotopes. They manually mapped VPHPTWMR to the known structure of actin [PDB: 1ATN] and suggested that it might correspond to residues: V129, P130, H101, P102, T358, W356, M355, R372[11]. In 2003, Mumey et al used FINDMAP to align VPHPTWMR to the actin sequence without utilizing information on the antigen structure. The result from FINDMAP shows VPHPTWMR can be mapped to residues as V129, P130, H101, P102, T103, W356, M355, R372[13]. FINDMAP mapped the input sequence to a slightly different set of residues (using T103 instead of T358). When running MIMOX with all parameters as defaults, we got no result. However, after the distance threshold is changed to 12 Å (the maximum distance allowed in MIMOX), we find that the two mappings above are returned as candidate cluster 5 and candidate cluster 17. As the side chain of some amino acids (such as arginine) can span a distance as great as 12 Å, MIMOX takes this value as the maximum allowable distance. This distance restriction is also used by Mapitope[16,17]. In this case, the need for the higher distance threshold is due to R372 which lies some distance from the other mapped residues. MIMOX also suggested other possibilities such as cluster 1(V96, P102, H101, P130, T358, W356, M355, R372) which has a bigger solvent accessible surface, and cluster 26 (V96, P98, H101, P102, T103, W356, M355, R372), which clearly has 3 sequential segments, i.e. VPHPT, WM, and R.

The second case is taken from work by Enshell-Seijffers[16]. They used monoclonal antibody 17b, which is against HIV gp120 envelope glycoprotein, to select a phage displayed random peptide library and got a set of 11 mimotopes. Analyzing the mimotopes with Mapitope, they suggest that the 17b epitope might consist of the following residues and segments: L111, LKPCVK (116–121), P124, VITQ (200–203), CPKV (205–208), RIK (419–421), I423, I424, K432, P437, P438. The structure of HIV gp120 envelope glycoprotein in complex with 17b has been solved [PDB: 1GC1] and 17b epitope has been recorded in the CED database as CE0058, which is composed of CK(119,121) + VTQAC(200,202–205) + RKQI(419,421–423) + KMYP (432,434,435,437). Among the 24 Mapitope predicted residues, 11 are contact residues of the 17b epitope. Using default parameters, MIMOX derived a consensus sequence, [LV] RP [LT] [KR] LRE [LP] [RT] X [-R], from 17b mimotopes. MIMOX finds no result matching the whole consensus sequence. Using LRLR, a fragment of the consensus sequence as the input sequence and running MIMOX in conservative match mode with other parameters as defaults, the top result is I423, K421, I420, and R419; the 4th result is V200, K121, L122, K432 and the 5th result is I423, K432, L122, and K121. Taking the top 5 results together, 13 residues are suggested by MIMOX and 6 of them are contact residues of the 17b epitope. In this case, Mapitope gives more complete and elaborate result.

The third case is taken from 3DEX[14]. LLTTNKD is a mimotope selected from a phage displayed random peptide library using HIV positive patients' IgG. Using 3DEX to map this mimotope to the HIV gp120 envelope glycoprotein [PDB: 1G9M], Schreiber et al reported that this mimotope might correspond to residues: L452, L453, T283, T455, N280, K282, and D279. When running MIMOX with default parameters, the top candidate residue cluster is exactly the same as the result of 3DEX, which has a 265.82 Ų solvent accessible surface. By picking candidate residues in conservative mode and clustering based on Cα atoms with a distance threshold 7 Å, the top candidate residue cluster suggested by MIMOX is changed to I360, I467, S465, T358, N356, K357, and E464. This cluster has a 399.28 Ų accessible surface area. Furthermore, when neighbouring based on all heavy atoms with a distance factor 1.11, the top candidate residue cluster suggested by MIMOX is L86, V85, T244, S243, N229, K231, and E267, which has an even larger accessible surface area, 562.89 Ų. All 3 clusters are shown in Figure ​Figure4.4. As the latter two mapping results suggested by MIMOX are more exposed, they might be able to bind to the antibody more easily.

The last case is taken from MIMOP[15]. BO2C11 is a human monoclonal antibody against human coagulation factor VIII. Villard et al selected two phage displayed random peptide libraries with BO2C11 and got a set of 27 mimotopes[26]. Very recently, Moreau et al have applied their newly developed tool MIMOP to analyze these mimotopes. Combining the two methods MimAlign and MimCons in MIMOP, the BO2C11 epitope is predicted be composed of a segment YFTNMF (2195–2200) and residues T2202, K2207, R2215, R2220, Q2222. The structure of human coagulation factor VIII in complex with BO2C11 has been solved [PDB: 1IQD] and the BO2C11 epitope has been recorded in the CED database as CE0176, which consists of FTNMF (2196–2200), R2215, RPQV (2220–2223), SLLT (2250–2253), HQ (2315–2316). Using default parameters, MIMOX derived a consensus sequence of [NQKR] [HST] RWSNRSS [ST] from those mimotopes. Again, the whole length consensus sequence returns no mapping results. However, when we use QH, RWSN, RSSS, three sequential fragments that cover the whole consensus sequence, as input sequences and running MIMOX in conservative match mode with all other parameters as defaults, the top 3 results of each of the partial sequences overlap with the MIMOP result and the native BO2C11 epitope well. For example, the third result of the input QH suggested by MIMOX is Q2316, H2315; the third result of the input RWSN suggested by MIMOX is R2220, F2196, T2197, N2198; the first result of the input RSSS suggested by MIMOX is R2215, S2216, T2202, T2197 and third result is K2249, S2250, S2254, T2253.

The programs in the second category include SiteLight, 3DEX, Mapitope, and MIMOX. SiteLight was implemented in C++ and it has been tested on Red Hat Linux. First, the program divides native protein surface into overlapping patches based on geodesic distances between residues; then aligns each mimotope in the library with each patch and scores and sorts them; finally, high scoring matches are selected iteratively until 25% of the native protein is covered[12]. Another program 3DEX was implemented in Visual Basic and could only run on Windows. It divides a sequence into a set of overlapping subsequences with a user-defined length (3-maximum length of mimotope). Then, it searches for matching residues at each position of the above subsequences against the sequence or PDB structure of native protein and links the neighbours iteratively until the first subsequence is complete. This is repeated for the following subsequences to complete the mimotope and return the result[14]. Mapitope was also implemented in C++ and its algorithm was first described by Enshell-Seijffers in 2003. Briefly, Mapitope deconvolutes a set of mimotope sequences into a set of overlapping amino acids pairs (AAP). Then a set of major statistically significant pairs (SSP) are identified based on the AAP. Later, the SSP are mapped and clustered in the antigen structure. Finally, the most elaborate and diverse clusters on the antigen surface are identified and regarded as the predicted epitope candidates[16,17].

MIMOP, a work published very recently, comprises the third category. MIMOP includes two approaches. One is called MimAlign, which can predict potential epitopic regions (PER) from mimotope and antigen sequences, and from the antigen structure if available. The other called MimCons, can predict PER from mimotope sequences but requires the antigen structure[15]. It seems that MIMOP can work with or without the antigen structure from the published case studies. However, the sequence of the only case that is independent of antigen structure is just a continuous subsequence of the antigen sequence. Thus, more studies are still needed to prove that MimAlign can work without antigen structure information.

All the existing programs described above have succeeded in given cases. However, a systematic evaluation on these tools is absent. Moreover, as shown in the Table ​Table1,1, most of the existing tools have not been implemented as a publicly accessible online service until now, making it less convenient for the community to access, utilize, and evaluate them.