Solid-Phase Synthesized Imunofan Confers Clinically Relevant Protection using a Chimeric Protein Vaccine Candidate Against E. coli O157:H7 in BALB/c Mice

Introduction/Objective: This study aimed to evaluate the efficacy of Imunofan, a synthetic peptide adjuvant, compared to Freund’s adjuvant. We hypothesized that imunofan would enhance protective immunity while avoiding the adverse effects associated with traditional adjuvants. Methods: Imunofan (836 Da) was synthesized via SPPS, purified by RP-HPLC, and validated by LC-MS. A chimeric antigen (ESI) encoding EspA, StxB, and Intimin was expressed in E. coli BL21(DE3) using the pET28-ESI plasmid, induced with IPTG, and purified via Ni-NTA chromatography. BALB/c mice (n = 10/group) were immunized with: (1) ESI+Imunofan, (2) ESI+Freund’s adjuvant, (3) ESI alone, or (4) Imunofan alone. IgG titers were measured by ELISA, and protection was assessed via bacterial shedding (log10 CFU/g feces) post-challenge with E. coli O157:H7. Results: ESI+Freund’s adjuvant elicited the highest IgG response (mean ± SEM: 12.3 ± 0.8 log10; p < 0.05 vs. ESI alone). Surprisingly, ESI+Imunofan showed a comparable reduction in bacterial shedding (3.1 ± 0.4 log10 CFU/g vs. Freund’s adjuvant: 2.9 ± 0.3; p > 0.1), despite lower IgG (9.1 ± 0.6 log10). ESI alone reduced shedding (4.2 ± 0.5 log10; p < 0.01 vs. control), outperforming Imunofan alone (5.8 ± 0.7; p < 0.05). discussion: Our results demonstrate that while Imunofan induces weaker humoral responses than Freund’s adjuvant, it provides comparable protection against E. coli O157:H7 shedding, suggesting its efficacy may rely on non-antibody-mediated immune mechanisms. These findings position Imunofan as a promising synthetic alternative to conventional adjuvants, particularly for vaccines where cell-mediated or mucosal immunity is critical. Conclusion: Imunofan’s structural authenticity and functional efficacy were demonstrated. Its comparable protection to Freund’s adjuvant, despite weaker humoral responses, suggests a unique role in modulating non-antibody-mediated immunity. These findings support imunofan as a safer alternative to conventional adjuvants.