Design, Synthesis and Primary Immunologic Evaluation of M2e-CRM197 Conjugate as a Universal Influenza Vaccine Candidate

Abstract

Background: Influenza is a contagious respiratory illness caused by an acute infection of influenza viruses, among which influenza A virus causes seasonal epidemic infections nearly every year. Due to the unpredictability of the evolving influenza A virus and time-consuming vaccine development cycles, novel universal influenza vaccines designed to induce broadly cross-reactive immune responses against frequently mutant influenza A virus strains are urgently required. Objective: The aim of this study was to synthesize a novel vaccine through the dual-site specific conjugation of the constant epitope of 23 amino acids (M2e) of the influenza A virus with a highly immunogenic carrier protein of Cross-Reacting Material (CRM197) under denaturation and to evaluate its primary immunogenicity in mice. Methods: The antigen (M2e) and the carrier protein (CRM197) were linked with different types of hetero-functionalized linkers, α-Maleimide-ε-Hydrazide Polyethylene Glycol 2k (MAL-PEG-HZ) and N-β-Maleimidopropionic Acid Hydrazide (BMPH) separately. The immunogenicity of the M2e-CRM197 conjugates with different types of linkers was evaluated in mice, and the M2especific total IgG and IgG-isotypes were determined by ELISA. Results: Immunogenicity studies revealed that anti-M2e antibody could be induced by the conjugate products, M2e-PEG-CRM197 and M2e-BMPH-CRM197, by approximately 30 and 90-fold higher than that of the M2e group. In addition, the anti-M2e antibody level induced by M2e-PEG-CRM197 conjugate was three times higher than that of M2e-BMPH-CRM197 conjugate, and the former could simultaneously activate both cellular and humoral immune responses. Conclusions: The M2e-CRM197 conjugated vaccines we synthesized in this study are highly immunogenic compared with M2e alone. Besides, evidence presented here indicated that the hydrophilic, non-immunogenic and biocompatible chain of the cross-linker might be a better choice for the development of a conjugate vaccine.