Bivalent mRNA-LNP vaccine against foot-and-mouth disease virus elicits cross-reactive immune response
Background: Physical biocontainment limitations of conventional inactivated virus vaccine platforms and genetic bottleneck of multi-serotype FMDV
Globally, inactivated vaccines for foot-and-mouth disease (FMDV) control necessitate strict BSL-3 facilities for large-scale production of high-risk pathogens and suffer from engineering limitations, including structural instability and deficient cross-immunogenicity of antigens. The rapid mutation rates of serotypes A and O have rendered conventional, linear, and static design approaches ineffective in providing cross-protection against interspecies transmission. Existing processes compromise the molecular integrity of antigens due to host-derived impurities and cell lysis-induced structural degradation during virus cultivation, a critical issue highlighted in vaccine stability research (DOI: 10.1016/j.vaccine.2023.05.012). Furthermore, the persistence of non-structural protein-derived impurities leads to critical false-positive results in diagnostic systems that differentiate vaccinated and infected animals, creating data bottlenecks in farm and national biosecurity governance. This distorts real-time feedback loops for effective vaccine concentrations and complicates batch effect control during commercial production by multinational pharmaceutical companies (e.g., Boehringer Ingelheim, Zoetis), contributing to major failures in large-scale disease control.
Discovery: In silico epitope screening and demonstration of cell-resolution Th1-GC B cell immune tensor synchronization
To overcome these data gaps, we developed a divalent mRNA@LNP design platform combining immunodominant epitopes from the VP1 structural protein of FMDV serotypes A and O with conserved T-cell epitopes from the 3A non-structural protein. In silico structural predictions were performed to optimize epitope-binding free energy, and codon optimization algorithms were used to maximize expression efficiency. In vivo mouse studies demonstrated that a 5-microgram mRNA@LNP dose elicited neutralizing antibodies comparable to those induced by conventional inactivated vaccines, while a 10-microgram booster dose induced Th1-biased immune cell activation that significantly exceeded the baseline of the ISA 206 oil-adjuvanted vaccine. Flow cytometry and single-cell omics analyses confirmed a dramatic increase in IFN-gamma levels and demonstrated multidimensional immune tensor synchronization of GC B cells and CTLs. These findings surpass recent advancements in LNP vaccine delivery research (DOI: 10.1038/s41587-024-02150-z) and represent a significant achievement in reorienting the topological variation curve of downstream transcriptomic networks towards immune induction.
VP1-3A heterologous epitope tuning and establishment of a reversible homeostatic precision stratification model
The mechanism of action involves the delivery of mRNA into target dendritic cells via highly structured LNP carriers, stimulating endogenous expression of the VP1-3A heterologous antigens. This process is characterized by a dramatic increase in the IgG2a/IgG1 ratio, a hallmark of humoral immunity, which precisely regulates CD8+ T cell activation. This platform enables the establishment of a precision stratification protocol based on omics matrix-based animal population immune genetic gradient mapping, overcoming inter-individual reactivity heterogeneity. By quantitatively upregulating or downregulating intracellular antigen concentrations through differential equations based on antigen translation rate kinetics, the model inhibits excessive inflammation and autonomously regulates the reversible transition to memory cells. This establishes a digital biosecurity control model that maintains high consistency in the homeostatic state of livestock populations within stressful microenvironments.
Outlook: Establishment of a programmable veterinary omics standard and implementation of a next-generation IND digital governance system
Demonstration of the divalent mRNA@LNP platform resets the animal vaccine R&D paradigm from a static, post-hoc, symptomatic approach to an AI-driven, multidimensional tensor-based, programmable molecular engineering backbone. A genetic gradient correction module was implemented to design and screen novel variant vaccine candidates within weeks during large-scale FMD outbreaks, virtualizing the screening process. In the global veterinary finished drug market (e.g., Merck Animal Health), this architecture establishes a computational moat for zero-variance between cGMP batches, addressing regulatory hurdles and serving as a critical business asset. Furthermore, it is expected to function as a digital healthcare governance backbone that meets companion diagnostic (CDx) biomarker panel requirements and disruptively shortens the approval timeline for the IND fast-track review framework of global regulatory agencies such as the US FDA and USDA, and the European EMA.
Foot-and-mouth disease (FMD) poses a significant threat to global livestock industries due to its high viral contagiousness. Despite inactivated viral vaccines remaining effective, their production requires stringent biocontainment and faces limitations in stability and cross-serotype immunogenicity. Here, we developed a novel bivalent mRNA@LNP vaccine targeting FMDV serotypes A and O. The design incorporated optimized immunodominant epitopes from the VP1 protein, combined with a conserved T-cell epitope from the 3A non-structural protein. We validated antigen expression in vitro and evaluated the immunogenicity of mRNA@LNP in mice. It elicited faster early immune activation and stronger humoral and cellular immune responses than the inactivated virus vaccine. Throughout the entire immunization period, the antibody levels induced by 5 μg mRNA@LNP were consistently equivalent to those of the 5 μg inactivated vaccine. Furthermore, the immune efficacy of the 10 μg mRNA dose following the booster vaccination was consistent to that of the ISA 206-adjuvanted inactivated vaccine. Liquid-blocking ELISA indicated that immunization of the mRNA@LNP induced strong blocking antibody titers against both serotype A(titer>256 at 5 μg) and serotype O(titer>128 at 10 μg). Antibody isotyping (high IgG2a/IgG1 ratio) and cytokine profiling (high IFN-γ) revealed a pronounced Th1-skewed immune response, consistent with enhanced GC B cell activation and CTL activation. In contrast, the ISA-206 adjuvanted vaccine induced a weaker cellular immune response and a more Th2-biased profile, highlighting the capacity of the mRNA vaccine to achieve not only comparable humoral immunogenicity to inactivated vaccines but also a more balanced and robust cellular immunity. These findings support mRNA vaccines as a promising, virus-free platform for multivalent FMD vaccines, offering potential solutions to current limitations and providing broader protection against FMDV outbreaks.
The divalent mRNA@LNP vaccine design demonstrated in this study goes beyond theoretical molecular immunological mechanisms and directly translates into practical applications for global finished drug supply chains and next-generation precision animal biotechnology businesses.
First, by instantly scanning the binding kinetics of FMDV VP1 structural protein and 3A non-structural protein using a Python algorithm in clinical settings, it eliminates the temporal noise caused by the immunogenicity deficiencies of conventional inactivated vaccines, safeguarding the exclusive immune protection barrier of livestock farming.
Simultaneously, by linking to open-source PDB and GenBank databases containing multidimensional protein genomic omics matrices, it enables the virtual simulation of immune escape variants during clinical trial design and the real-time reverse calculation of effective docking concentrations for target neutralizing epitopes, realizing a companion diagnostic (CDx) panel interface.
Furthermore, by linking neutralizing antibody titers and GC B cell activation induction indicators as correction factors during large-scale clinical trials of multinational companies' next-generation multivalent FMD mRNA vaccines, it eliminates inter-batch variations in neutralizing capacity and maximizes the probability of obtaining clinical trial protocols and cGMP commercial operation approvals from global regulatory agencies, serving as a backbone infrastructure.