Characterizing Complex Polysera Produced by Antigen- Specific Immunization through the Use of Affinity- Selected Mimotopes Galina Denisova, Dimitri Denisov, Carole Evelegh, Michaela Weissgram, Jochen Beck, Stephen Ronan Foley, Jonathan Lorne Bramson* Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Abstract Background: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. Methodology/Principal Findings: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. Conclusions/Significance: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines. Citation: Denisova G, Denisov D, Evelegh C, Weissgram M, Beck J, et al. (2009) Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes. PLoS ONE 4(4): e5309. doi:10.1371/journal.pone.0005309 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received January 25, 2009; Accepted March 16, 2009; Published April 23, 2009 Copyright: 2009 Denisova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the CBCRA (TAGS grant #14450; www.breast.cancer.ca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: bramsonj@mcmaster.ca Introduction Proper characterization of the antibody response produced by vaccination is dependent upon the ability to characterize the specific target epitopes that are recognized by the polyserum. As an example, the HER-2 protein is over-expressed in ,30% of breast cancer patients and is associated with poor prognosis [1,2] making HER-2 an attractive target for the development of antigen-specific cancer vaccines. Caution is needed, however, as only antibodies to specific epitopes can suppress tumor growth [2,3] while others actually stimulate growth [4]. As such, measurement of total levels of antigen-specific antibody is not an appropriate measure of vaccine activity since not all antibodies would be able to provide therapeutic effect. Rather, it is necessary to employ assays that permit quantification of antibodies to specific functional epitopes to adequately monitor B cell immunity. Given the broad range of potential epitopes within a protein, characterizing the breadth of epitopes recognized by a particular polyclonal response is quite challenging. While it may be possible to monitor antibodies directed against linear epitopes using synthetic peptides, the majority of antibody responses [5] are directed at structural epitopes which are difficult to recapitulate with synthetic peptides. We have been investigating methods that employ affinity- selection of random peptides to provide an unbiased approach to epitope characterization. We have previously demonstrated that this method can be used to characterize the target epitopes of monoclonal antibodies [6,7,8,9,10,11,12,13,14] and, more recently, we have also applied this technology to polyclonal serum [15]. Manipulation and identification of random peptides is facilitated by the use of phage-display [16] where the random peptides presented on the phage surface serve as peptidomimetics of conformational and discontinuous antibody epitopes [17,18] referred to commonly as ‘‘mimotopes’’. Isolation of specific mimotopes is accomplished through affinity selection where antibodies of interest are first bound to a solid matrix and subsequently adsorbed to the phage library expressing the mimotopes. Iterative washing and adsorption steps allows for enrichment of phage carrying mimotopes which are specific for the bound antibodies. The mimotope sequences are then analyzed and assigned a location on the target protein using algorithms that we have designed [9,15]. In the current manuscript, we have applied this method to characterize the specificity of polyclonal responses produced following immunization against a specific antigen. We have chosen HER-2 as a model antigen. As stated previously, this protein is an interesting target for vaccine development but the diverse biological effects of individual HER-2 antibodies (either stimulatory or inhibitory) necessitate careful analysis of the polyclonal repertoire following immunization. Epitope selection PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5309