Multi‐Architecture Nanovaccines Against Pseudorabies Virus and Particle Size‐Governed Immune Activation Mechanisms

ABSTRACT Pseudorabies virus (PRV), characterized by latent infection, lifelong viral shedding, and high mortality rates, has inflicted substantial economic losses on the swine industry. While vaccination remains the most cost‐effective control strategy, emerging virulent variants with enhanced immune evasion capabilities have compromised conventional vaccines, manifesting as short‐lived protection, suboptimal efficacy, and risks of latency reactivation. To overcome rapid antigen clearance and undefined immunomodulatory mechanisms in traditional emulsion vaccines, we engineered three nanoemulsions with distinct architectures and sizes: an oil‐in‐water nanovaccine (O/W nanovaccine, 163.1 ± 3.84 nm), a water‐in‐oil‐in‐water nanovaccine (W/O/W nanovaccine, 32 ± 4.04 nm), and a Pickering emulsion nanovaccine (326.07 ± 9.19 nm). The immunogenicity and biocompatibility of nanoemulsion formulations are systematically evaluated through in vitro cellular models, followed by comprehensive in vivo investigations in murine/porcine models to elucidate immune mechanisms, protective efficacy, and challenge resistance. All formulations demonstrated immunostimulatory potential with distinct functional advantages. O/W nanovaccine exhibited superior antigen‐presenting cell uptake efficiency and sustained cytokine induction. W/O/W nanovaccine showed maximal dendritic cell activation and high‐titer neutralizing antibodies. Pickering emulsion nanovaccine enhanced specific antibody titers. In addition, mechanistic studies revealed that nanoscale lymphatic targeting of O/W nanovaccine and W/O/W nanovaccine leveraged immune cell size preferences for polyfunctional cytokine release. Multi‐layered design of W/O/W nanovaccine enabled compartmentalized antigen delivery, induced CD8+ T cell response, and synergistically enhanced cross‐presentation to elicit coordinated humoral and cellular immunity. Particulate‐stabilized interface of Pickering emulsion nanovaccine enhanced humoral immunity via DC‐mediated IFN‐γ hyper‐secretion and CD4+ T cell differentiation. Furthermore, all nanovaccines demonstrated higher protective efficacy compared to commercial vaccines in animal challenge models infected with PRV, O/W nanovaccine achieved 100% survival in mice while exhibiting the lowest viral shedding in pigs. This study establishes a transformative prevention paradigm against PRV through nanovaccine engineering, providing critical insights for developing next‐generation veterinary vaccine platforms.