by antigen specific B cells will be influenced by the antigen structure within the vaccine inoculum. As such, the diversity of the polyclonal antibody response is a direct reflection of the specific vaccine formulation. To determine the utility of our mimotope technology for characterizing the epitope diversity of different polysera directed against the same protein, we examined the binding properties and epitope specificity of mouse polyserum against HER-2 generated by different vaccine inocula. Results and Discussion The composition of polyclonal responses generated in response to rat HER-2 is dependent upon the method of immunization and structure of the antigen Mice were immunized against rat HER-2 using different strategies to determine how the method of exposure influences the antibody response. We chose to employ a xenoantigen, rat HER-2, rather than the murine homolog because our previous experience has determined that xenoantigens are more immuno- genic than native antigens and yield cross-reactive immune effectors that recognize both the native and xenoantigen [19,20,21]. We opted to employ plasmid vaccines expressing different forms of HER-2 since this method has been shown to be highly effective in mice [22,23,24]. Previous work has found that similar titers of antibodies against the HER-2 extracellular domain (ECD) were evoked by immunization with plasmids expressing either the soluble ECD or full length HER-2 [24]. Given the likelihood that soluble ECD and membrane-bound ECD would adopt distinct structures and give rise to a different spectrum of antibodies, we sought to compare the properties of polysera produced by plasmid immunization with the 2 forms of HER-2. To this end, mice were immunized with plasmids expressing either the full length protein (HER-2FL) or the soluble extracellular domain only (HER-2ECD). We also observed that mice inoculated with TUBO tumors, which express rat HER-2, developed high titer antibodies against rat HER-2 (data not shown). Since the structure of the full-length protein on tumor cells may also be different than full-length protein expressed following plasmid vaccination, we included this polyserum in our analysis, as well (HER-2TUBO). All 3 methods yielded similar titers of antibody as determined by ELISA (Fig. 1A). Using Western blot analysis, we observed that the polyserum generated in response to HER-2ECD recognized both native and denatured forms of HER-2 equally well (Figure 1B). By contrast, binding of HER-2 by HER-2FL and HER-2TUBO polyserum was completely ablated by denaturation (Figure 1B). Since it appeared that the antibodies generated in mice exposed to full-length HER-2, either through plasmid vaccination or exposure to tumor cells, have distinct specificity relative to the antibodies produced in response to HER-2ECD, we examined the ability of the sera to recognize native HER-2 expressed on the surface of TUBO cells. For this experiment, TUBO cells were coated with various dilutions of serum and the quantity of bound antibody was determined by flow cytometry. Unfortunately, we could not employ this method to study polyserum produced by TUBO cells themselves because we could not distinguish HER-2 specific antibody from other TUBO- specific antibodies. Nonetheless, these data demonstrate that although the HER-2ECD and HER-2FL polysera exhibit compa- rable titers of anti-HER-2 antibody by ELISA (Figure 1A), the HER-2FL polyserum had markedly enhanced capacity to bind the native protein (Figure 1C), suggesting that the polyserum produced by HER-2FL may be more specific for the native form of the protein. Given the observation that the HER-2ECD Epitope Mapping with Mimotopes polyserum was less sensitive to protein structure, we hypothesized that this polyserum would also prove to be more cross-reactive. Indeed, when the various polysera were tested for cross-reactivity by Western blot, to another form of HER-2 (human HER-2), the HER-2ECD polyserum revealed some cross-reactivity whereas the HER-2FL and the HER-2TUBO showed no binding to the human protein (Figure 1 D). Thus, although all 3 polyserum exhibit similar titers of anti-HER-2 antibodies by ELISA using native protein, it is clear that each polyserum is composed of a discrete combination of individual antibody specificities. Differential polyserum compostion correlates with impact on tumor growth in vitro To gain further insight into possible differences in the polysera, we tested whether the anti-HER-2 antibodies would suppress TUBO cell proliferation in vitro. TUBO cells were grown for 2 weeks in the presence of IgG purified from the various polysera (25 mg/ml final). Only IgG purified from the HER-2FL serum suppressed tumor cell growth whereas the other sera had no effect (Figure 1E; p,0.01 based on ANOVA and post-hoc Tukey test). These results again demonstrate that the polyserum produced by the 3 immunization methods have different functional properties. Notably, even though both sera produced in response to full-length protein (HER-2FL and HER-2TUBO) display similar dependence upon protein structure for binding, the functional effects of these polysera appear to be distinct. This difference may reflect differential abundance of inhibitory and activating antibodies. Characterization of eptiope specificity within the anti- HER-2 polysera using affinity-selected random peptides Characterization of epitope specificity for antibodies can be quite challenging, particularly in the case of structural epitopes. Since binding of both HER-2FL and HER-2TUBO sera to recombinant HER-2 is abrogated when the protein is denatured, it would appear that the epitopes recognized by these sera are largely conformational. Therefore, to characterize the epitope- specificity of these sera, we opted to employ a mimotope-based strategy where random circularized peptides displayed on recombinant phage are affinity-selected using specific polyserum and a computer algorithm is employed for epitope prediction based on the sequence of the selected mimotopes [9,15]. To facilitate the manipulation and identification of these random peptides, we have employed a phage-display method where random 10-mer circularized peptides are expressed on N-terminus of the phage major coat protein. Since unrelated antibodies present in mouse serum can confound our analysis, the phage library was adsorbed to na¨ıve mouse serum prior to further analysis to remove all phages that were reactive with general mouse antibodies. Likewise, anti-phage antibodies present in mouse serum would also complicate interpretation, so the sera to be tested was adsorbed to immobilized wild type phage fd-tet [25] to remove phage-specific antibodies. Phage bearing specific mimotopes were enriched through iterative immunoaffinity selection steps on immobilized anti-mouse IgG. Specific phages were defined as those which bound to serum from immunized mice but not to serum from na¨ıve mice. The sequences of the specific mimotopes selected through iterative rounds of screening are presented in Table 1. Interestingly, we did not find any common mimotopes in the collection of sequences selected by each individual polyserum suggesting that each serum is composed of distinct antibodies. It is also notable that we only uncovered a limited set of phages with HER-2TUBO serum, suggesting that this polyserum has a limited range of specificity. PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5309