Heat-stable mRNA vaccine platform to protect farmed fish against Vibrio harveyi infection
Background: Structural limitations of existing inactivated/recombinant subunit vaccine systems and bottlenecks in antigen presentation/cold chain data for marine aquaculture vibriosis R&D.
Existing marine aquaculture disease prevention strategies have relied on formalin-inactivated whole-cell vaccines or recombinant protein subunit injections. However, this linear and static analytical standard guideline exposes critical vulnerabilities. The major virulence factor of Vibrio harveyi, FlaA flagellin, exhibits optimal TLR5 receptor binding free energy only in its native conformation. During the inactivation process, thermal denaturation and chemical cross-linking irreversibly disrupt the epitope conformation, resulting in a 60-80% reduction in neutralizing antibody titers compared to baseline, creating a cellular dissociation-induced structural noise. The large yellow croaker (Larimichthys crocea) aquaculture industry exceeds 250,000 tons in annual production in China alone, and mortality rates due to vibriosis can reach up to 70% in high-density aquaculture farms, yet there are no commercial vaccines available. Antibiotic overuse accelerates the horizontal transfer of multidrug resistance (MDR) plasmids in oxytetracycline- and florfenicol-resistant V. harveyi strains, and the accumulation of residual antibiotics in the marine ecosystem violates EU Regulation 2019/6 and Codex Alimentarius MRL standards, potentially disrupting export supply chains. Furthermore, maintaining the cold chain for aquatic vaccines, which requires 2-8°C refrigerated logistics, is impractical in Southeast Asia and Africa, where aquaculture is prevalent, reducing actual vaccination rates to below 20%. This infrastructure bottleneck has been a fundamental barrier to achieving effective seroconversion.
Discovery: FlaA mRNA-LNP microfluidic encapsulation and single-cell resolution renal tissue transfection tensor synchronization demonstration.
In this study, the full-length ORF of V. harveyi FlaA flagellin was codon-optimized (based on the Codon Adaptation Index for species-specific tRNA pools), and the 5′-UTR/3′-UTR was dually optimized to modulate the binding free energy of the translation initiation complex (eIF4F). CleanCap AG technology-based co-transcriptional capping achieved a 95.1% capping efficiency, significantly exceeding the 70-85% efficiency of conventional enzymatic post-transcriptional capping. The lipid nanoparticle (LNP) formulation was optimized using a system of differential equations-based particle self-assembly rate constant in silico pre-calculation for the molar ratio of ionizable lipid, helper lipid, cholesterol, and PEG-lipid. The microfluidic rapid mixing (staggered herringbone mixer) process eliminated batch effects, resulting in a homogeneous nanoparticle population with a polydispersity index (PDI) of less than 0.1. After 12 hours of renal cell transfection, the mRNA residual rate was greater than 50%, indicating that the LNP's endosomal escape efficiency and mRNA double-strand secondary structure stability are sufficiently maintained, even under conditions where RNase activity in fish cells is 2-3 times higher than in mammalian cells. After lyophilization, the physicochemical parameters (particle size, zeta potential, encapsulation rate) and in vitro antigenicity were maintained at 25°C for 10 weeks, demonstrating a thermostable profile. This represents the first extension of the room-temperature stability benchmark achieved by Moderna's mRNA-1283 (second-generation COVID-19 vaccine) lyophilized formulation to the aquatic vaccine field, revealing a downstream transcriptome network topological variation curve.
Following intramuscular injection, robust expression of FlaA protein in muscle tissue, elevated levels of specific IgM and neutralizing antibodies, increased activity of antioxidant enzymes (SOD, CAT) and metabolic enzymes (ALP, LZM), and upregulation of immune-related genes (IL-1β, TNF-α, MHC-II, IgT) in immune tissues (spleen, kidney, liver) were observed, demonstrating the molecular integrity of humoral and cellular dual immune responses.
FlaA-TLR5 axis immune signaling pathway modulation and establishment of a precise layered model for reversible fish mucosal/systemic immune homeostasis.
V. harveyi challenge test results showed a survival rate of 77.7% and a relative percent survival (RPS) of 70.1% in the 20 μg administration group, meeting the EU aquatic vaccine efficacy criterion of RPS ≥60% and significantly exceeding existing inactivated whole-cell V. harveyi vaccines (RPS range of 30-50%). Omics matrix-based analysis revealed a nonlinear dose-response curve for immune responses at different doses, and 20 μg was identified as the optimal dose, precisely reaching the saturation threshold of the MyD88-NF-κB downstream signaling cascade after FlaA-TLR5 binding, representing the rate-limiting step. This precision stratification model enables molecular phenotype clustering based on species-specific body weight, immune maturity, and aquaculture water temperature/salinity gradients, and provides a reversible autonomous modulation backbone for effective neutralizing antibody titers even under aberrant environmental stress by up- or down-regulating the rate-limiting step constant in high-temperature/high-density stress and low-temperature/low-density environments. Furthermore, the modularity of the mRNA platform allows for the immediate design of multivalent vaccines containing additional antigen ORFs, such as VhP (V. harveyi hemolysin) and OmpK (outer membrane protein), which contrasts with the single-antigen limitation of existing subunit vaccines from Elanco Animal Health and MSD Aqua.
Outlook: Establishing a standard for programmable aquatic precision immunology and launching a digital governance system for next-generation aquatic biopharmaceuticals.
This study represents a declarative turning point in the governance of aquatic disease R&D, shifting from a static, post-hoc, symptomatic antibiotic-based system to a fully reset, AI-driven, multidimensional tensor-based programmable mRNA vaccine infrastructure. The global aquatic vaccine market is projected to grow rapidly from approximately $480 million in 2025 to $820 million in 2030 (CAGR ~11.3%), and the mRNA platform is poised to emerge as the next-generation backbone, following DNA vaccines (Elanco APEX-IHN, the world's first aquatic DNA vaccine) in this market. The lyophilized room-temperature stabilization technology explicitly establishes a computational moat by linking species-specific codon optimization index and LNP ionizable lipid pKa values as correction factors in high-throughput mass vaccination screening in regions with poor cold chain infrastructure (e.g., Indonesia, Bangladesh, Egypt), thereby zeroing out batch-to-batch variation in encapsulation rate and particle size. The microfluidic continuous process is directly compatible with the Precision NanoSystems (now Cytiva) NanoAssemblr platform, and GMP scale-up can achieve a coefficient of variation (CV) of less than 5% between batches. Furthermore, this platform can be rapidly expanded to include other major aquatic pathogens, such as Aeromonas hydrophila, Edwardsiella piscicida, and Streptococcus iniae, and introduces the concept of digital companion diagnostics (CDx) to the aquaculture field—real-time metagenomic monitoring of pathogens in aquaculture farms and automated updating of mRNA vaccine antigen sequences—to disrupt the OIE (WOAH) Aquatic Animal Health Code and national regulatory frameworks for aquatic biopharmaceutical IND approval timelines, creating a master asset.
When entering the Category I New Veterinary Drug registration pathway for aquatic veterinary drugs in China, the platform's room-temperature stability, high RPS, and modular antigen replacement capability will be decisive differentiating factors that meet the requirements for expedited review.
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.
The development of the mRNA-LNP thermostable fish vaccine platform in this study goes beyond theoretical aquatic immunology and directly translates into the global supply chain of finished aquaculture products and the next generation of precision aquaculture bio-businesses.
First, by instantly scanning the in vivo kinetics of V. harveyi FlaA-specific neutralizing antibody titers using a Python-based ELISA quantitative algorithm in aquaculture farms, the temporal noise of acute vibriosis outbreaks can be eliminated at the source, safeguarding economic losses at the farm level.
At the same time, by linking to open-source virulence factor databases aggregated in NCBI Protein, UniProt, and VFDB, the design of vaccines can virtually simulate and mitigate confounding variables of spurious cross-reactive epitopes, and a companion diagnostic (CDx) panel can be realized that allows for real-time reverse calculation of the effective TLR5 docking concentration of FlaA monomers.
Furthermore, when conducting large-scale clinical trials for next-generation aquatic pathogen-targeted mRNA therapeutics by multinational aquaculture bio-companies, by linking species-specific codon adaptation index and LNP ionizable lipid pKa values as correction factors, batch-to-batch variation in encapsulation rate and particle size can be eliminated, and the probability of obtaining OIE/WOAH and national regulatory agency approval for aquatic biopharmaceutical clinical trial protocols and cGMP commercial production can be maximized, creating a backbone infrastructure.