Previously, we designed a subunit vaccine candidate based on three L. intracellularis antigens with promising results in pigs. In this study, antigens were produced individually to achieve an even antigen ratio in the formulation. The emulsion characterization included the drop size and the mechanical and thermal stability. Immune response was evaluated by indirect and sandwich ELISAs, qPCR, and flow cytometry. The vaccine candidate’s safety was assessed by histopathology and monitoring the clinical behavior of animals. The average production yielded for the chimeric antigen as inclusion bodies was around 75 mg/L. The formulation showed mechanical and thermal stability, with a ratio Hu/Ho > 0.85 and a drop size under 0.15 nm. Antigens formulated at a ratio of 1:1:1 induced a significant immune response in inoculated pigs that persisted until the end of the experiment (week 14). The dose of 200 μg significantly activated cellular response measured by transcriptional and translational levels of cytokines. The cell proliferation assay revealed an increment of lymphocytes T CD4+ at the same dose. Animals gained weight constantly and showed proper clinical behavior during immunization assays. This research demonstrated the immunological robustness of the new subunit vaccine candidate against Porcine Proliferative Enteropathy evenly formulated with three chimeric antigens of L. intracellularis. © 2023 by the authors.