Development and immunoprotective evaluation of a thermostable mRNA vaccine targeting Vibrio harveyi flagellin in the large yellow croaker (Larimichthys crocea)

Developed a thermostable flaA mRNA-LNP vaccine against V. harveyi for large yellow croaker. • Stable at 25 °C for 10 weeks; eliminates cold-chain dependence. • High capping efficiency and antigen expression ensure strong immunogenicity in fish. • Elicits robust humoral and cellular immunity via the TLR5/MyD88 pathway. • Most effective dose among those evaluated: 20 μg, yielding 77.7% survival and 70.1% RPS. Vibrio harveyi causes severe vibriosis in the large yellow croaker ( Larimichthys crocea ), resulting in high mortality rates and significant economic losses in mariculture. Currently, there is no effective commercial vaccine available, and the overuse of antibiotics raises concerns about resistance and the environment. In this study, we developed a thermostable mRNA-LNP vaccine that encodes the FlaA flagellin, which is a key virulence and immunogenic antigen of V. harveyi . Through the optimisation of UTRs, capping, lipid nanoparticle (LNP) formulation, microfluidic encapsulation and lyophilisation, the vaccine achieved a capping efficiency of 95.1% and retained over 50% of the mRNA in kidney cells 12 h after transfection. The lyophilised vaccine remained stable with respect to physicochemical parameters and in vitro antigenicity at 25 °C for 10 wk, and retained the ability to induce neutralising antibodies. Intramuscular injection of the vaccine into croakers resulted in robust FlaA expression in muscle tissue, elevated levels of specific IgM and neutralising antibodies, increased antioxidant and metabolic enzyme activities, and expression of immune-related genes in immune tissues. A V. harveyi challenge demonstrated that a 20 μg dose was the most effective among the tested doses, with 77.7% survival and 70.1% relative percent survival. This is the first room-temperature-stable mRNA-LNP vaccine against an aquatic bacterial pathogen, offering a novel, eco-friendly strategy for controlling V. harveyi and advancing the development of mRNA vaccines for bacterial diseases in aquaculture.