Harnessing T4 phage-based platform for efficient design of ready-to-use multiepitope vaccines

Vaccine design strategies that elicit robust immunological responses still inadequately addressed in the era of emerging infectious diseases. To tackle this unmet challenge, we developed a T4 phage-based modular vaccine platform tailored for the rapid and efficient development of ready-to-use multiepitope vaccines. In the present study, a proof-of-concept study was conducted using Newcastle disease virus (NDV) that poses severe threats to the global poultry industry as an example. Initially, conserved neutralizing epitopes (designated as HN2) were selected from the hemagglutinin-neuraminidase (HN) protein of NDV using comparative epitope profiling. Next, HN2 was fused in triplicate with the small outer capsid (Soc) protein of T4 phage, generating a novel chimeric antigen Soc-3HN2. After conformation of its immunogenicity in vivo, the chimeric antigen was then immobilized on T4 phage via Soc-mediated conjugation, followed by in vivo assessment of its vaccine efficacy. Finally, the triplicated epitope (3HN2) was inserted downstream of the Soc-encoding sequence of T4 phage using CRISPR-Cas12a, enabling constitutive antigen display on the surface of engineered phage virions. The resulting T4 phage-based ready-to-use vaccine, 3HN2-T4, conferred complete protection in chickens against lethal challenge with the virulent NDV strain F48E9. Notably, 3HN2-T4 significantly reduced viral shedding and mitigated pathological lesions in infected chickens, with efficacy comparable to that of a commercial NDV vaccine. This study validates the T4 phage as a ready-to-use, highly versatile platform that supports flexible and efficient epitope vaccine design. Furthermore, it provides a generalized framework to accelerate the development of broad-spectrum multiepitope vaccines against diverse infectious pathogens.