Table 2. Occurrence of statistically significant amino acids within mimotope collections. HER-2ECDa mimotopes HER-2FL mimotopes HER-2TUBO mimotopes Residue Frequencyb Residue Frequency Residue Frequency Glu Pro Ser Ala Arg Thr His Asn Met Asp Gly Cys Val Gln Leu Trp Lys Tyr Phe Ile 10.07% 9.06% 8.05% 7.72% 7.38% 7.05% 6.38% 6.38% 5.70% 4.36% 4.03% 3.36% 3.69% 3.69% 3.02% 3.02% 2.01% 2.01% 1.68% 1.34% Gly Thr Pro Ser Ala Glu Arg Asp Trp Lys Gln Val Leu Asn Cys Phe Tyr His Met Ile 10.29% 9.88% 9.05% 8.23% 7.00% 7.00% 5.35% 5.35% 5.35% 4.94% 4.53% 4.12% 3.29% 2.47% 2.47% 2.47% 2.47% 2.06% 2.06% 1.65% Ser Pro Glu Ala His Thr Gly Asn Val Arg Tyr Gln Lys Phe Met Asp Cys Ile Leu 15.00% 13.75% 8.75% 8.75% 6.25% 6.25% 5.00% 5.00% 5.00% 3.75% 3.75% 3.75% 3.75% 2.50% 2.50% 2.50% 1.25% 1.25% 1.25% aPolyserum used for mimotope isolation. bThe frequency was determined as: (# specific residues/total # of residues in the collection) 6100. doi:10.1371/journal.pone.0005309.t002 HER-2 due to replacement T105 in the mouse protein with A105 in the rate protein). Common clusters ECD#3 and FL#3 are located within domain 3 around replacements of D363 for N and Q375 for H in the mouse protein. Cluster FL#4 is located on domains 1 and 3 close to replacement of R71 for H and close to cluster ECD#4 (close to replacements H457 for N). Cluster ECD#5 is located at the beginning of domain IV around replacement of D482 for A in mouse protein. Cluster FL#2 is located on loop 168–182 of domain 1 close to replacement of A177 for T. Thus, similar to T cell epitopes, B cell epitopes in xenoantigens may also be generated by specific non-conserved amino acids within the vicinity of the epitope which enables the B cells to overcome tolerance. Conformational analysis reveals distinct epitopes within similar regions It is interesting to note that although many of these epitopes map to similar regions of the protein, for the most part, they involve distinct amino acid residues (Table 4). We have observed similar outcomes using monoclonal antibodies directed against the West Nile virus E protein [9]. The differences in epitope selection noted in this manuscript probably reflect differences in confor- mation of the soluble and cell-attached proteins which alter the way the protein is bound by antigen-specific B cells. We found that most of the clusters are located at protruding loops or b-turns exposed on the protein surface as it was shown for many other epitopes [26]. Of interest, epitopes ECD#3 and FL#3 overlap Epitope Mapping with Mimotopes and involve many, but not all, of the same residues. These 2 clusters are located at two closely positioned turns connecting two b-strands and two anti-parallel a-helices. It is also interesting that clusters ECD#6, FL#5 and TUBO#1 are all located within the area of the epitope identified for pertuzumab [27], a therapeutic antibody currently under development. The pertuzumab epitope encompasses three loops. ECD#6 and TUBO#1 include the first loop of the pertuzumab epitope while cluster FL#5 includes the two adjacent loops. Although TUBO#1 overlaps with ECD#6, these two clusters only share 3 common residues. It is also striking that the HER-2 specific antibodies that were generated in response to growing TUBO tumors seemed to identify only a single cluster which is consistent with the limited number of mimotopes isolated by affinity selection with the HER-2TUBO polyserum (Table 4). It is also notable that overlapping clusters FL#4 and ECD#4 are planar surfaces formed by b-sheets and located close to each other in domain III; however FL#4 is shifted in the direction of domain I and is actually located at the interface between domain I and domain III. Antibody targets are defined by physico-chemical and geomet- rical properties such as hydrophilicity [28], chain flexibility [29,30], surface accessibility [38] and protrusion from the surface [31]. Changes of antigen conformation and its participation in various complexes should lead to corresponding changes in immune response [32,33,34]. Our results indicate that the method of immunization has a pronounced effect on the breadth of the antibody response against a given protein. Affinity-selected mimotopes can be used as a tool for characterizing these differences and providing a ‘‘signature’’ of the polyclonal response. Using predictive tools, this signature can also be used to characterize the specific binding sites for the antibodies which can offer insight into their functionality. Through iterative application of this technology in human studies of antibody- mediated resistance, it should be possible to develop ‘‘resistance’’ signatures which would be indicative of effective vaccination. Additionally, knowledge of mimotopes associated with specific ‘‘functional’’ domains may serve as the basis for developing epitope-specific vaccines. Methods Plasmid vaccines and immunizations The extracelluar domain of rat HER was amplified from cDNA using the primers 59GTCGAAGCTTATGGAGCTGG- CGGCCTGG and 59 GACTGAATTCTTAGTTGATGGGG- CACGG and inserted into the vaccination plasmid pcDNA3.1- 002 [35] to yield prHER-2ECD. Full length rat HER-2 was prepared by PCR amplification of full length HER-2 gene using primers 59-GCCCGGTGACATAAATCATTGCAACTGTAG and 59-CTACAGTTGCAATGATTTATGTCACCGGGC and inserted into pcDNA3.1-002 to yield prHER-2FL. Balb/c mice (6– 8 weeks old) were immunized by intramuscular injection followed by electroporation [36] with 100 mg of pHER-2ECD or pHER-2FL. One month later, the immunization was repeated. Serum antibodies were measured 2 weeks following the booster immunization. All animal work described in this manuscript was approved by the Animal Research Ethics Board of McMaster University. TUBO breast cancer tumor model The TUBO cell line, kindly provided by Dr. Guido Forni (Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy), was derived from a spontaneous mammary tumor which arose in a BALB NeuT transgenic mouse expressing PLoS ONE | www.plosone.org 6 April 2009 | Volume 4 | Issue 4 | e5309