No more than three PlpE non-overlapping epitopes trigger significant antibody production in individuals vaccinated with the <i>Pasteurella multocida</i> epitope-chimeric proteins

Current vaccine research still confronts multiple challenges, and epitope-focused vaccine design serves as an effective technical approach to address these issues. Antigens harbor multiple epitopes; however, the number of epitopes capable of triggering significant antibody responses in vaccinated individuals remains undefined. This study aimed to determine the number of non-overlapping epitopes-designated "effective epitopes" hereafter-that trigger significant antibody production in vaccinated individuals when presented in distinct PlpE epitope-chimeric proteins. Herein, chimeric proteins incorporating varying numbers of PlpE non-overlapping B-cell epitopes were generated. By analyzing serum antibody responses in vaccinated individuals, we defined the quantitative characteristic of PlpE-derived effective epitopes. The total number of PlpE effective epitopes in vaccinated individuals was 2.6 (95% CI: 1.909-3.201) for full-length PlpE and 1.2 (95% CI: 0.5426-1.857), 0.9 (95% CI: 0-1.820), and 0.2 (95% CI: 0-0.5016) for the PlpE chimeric proteins (PlpE-VP60P, PlpE-BcfA, PlpE-PtfA), respectively. With an increase in the number of PlpE non-overlapping epitopes incorporated into the chimeric proteins, the number of PlpE effective epitopes exhibited a trend of initial increase followed by a decrease. Ultimately, the average total number of effective epitopes across all PlpE chimeric proteins did not exceed 3, with the highest number 2.1 (95%CI: 1.572-2.628). In conclusion, the number of PlpE non-overlapping epitopes on an antigen, that trigger significant antibody responses in each vaccinated individual, is very limited.IMPORTANCEEpitopes underpin the antigenicity of protein antigens. Although the concept of antigenic epitopes has been proposed over 50 years, our understanding on epitopes remains incomplete. Multiple antigenic epitopes can be identified on a single antigen, while the number of these epitopes that function in vaccinated individuals remains unclear-a gap hindering the rational design of vaccines. In previous studies, we identified six non-overlapping epitopes of <i>Pasteurella multocida</i> PlpE. Herein, we found that the total number of non-overlapping epitopes-capable of significantly triggering antibody production-that are present in PlpE chimeric proteins does not exceed three per vaccinated individual. This finding offers important insights for rational vaccine design: given the highly limited number of non-overlapping epitopes that function in vaccinated individuals, only a limited number of epitopes can be grafted onto scaffold proteins. Epitope-focused vaccine design must, therefore, account for competitive interactions between epitopes on the new antigen.