A self-adjuvant trivalent T4 bacteriophage nanoparticle vaccine induces broad protective immunity against Orthoebolaviruses.
Orthoebolaviruses, including Zaire virus, Sudan virus, and Bundibugyo virus, remain major public health threats because of their high case-fatality rates and recurrent outbreaks. Although licensed vaccines provide effective protection against EBOV, broadly protective vaccines against multiple pathogenic orthoebolavirus species remain unavailable. Here, we developed T4 bacteriophage-based nanoparticle vaccines displaying glycoproteins (GPs) from EBOV, SUDV, and BDBV by leveraging the SpyTag/SpyCatcher conjugation system. Monovalent vaccine candidates and a trivalent formulation (T4-Mix) were generated to evaluate their potential for broad orthoebolavirus immunization. Intramuscular immunization of BALB/c mice elicited robust GP-specific humoral and cellular immune responses without the need for exogenous adjuvants. In homologous surrogate challenge models using replication-competent recombinant vesicular stomatitis viruses expressing EBOV, SUDV, or BDBV GP, all monovalent vaccines conferred complete protection against their respective challenge viruses. Furthermore, the optimized trivalent T4-Mix formulation provided complete protection against rVSV-EBOV, rVSV-SUDV, and rVSV-BDBV challenges. Collectively, these findings demonstrate that the T4 bacteriophage functions as both an antigen delivery vehicle and an intrinsic immunostimulatory scaffold, highlighting its potential as a versatile platform for the development of broadly protective multivalent orthoebolavirus vaccines.