Foot-and-mouth disease (FMD) is one of the major animal infectious diseases in the world. All cloven-hoofed animals are susceptible to FMD. Vaccination is still the first choice for the prevention and control of FMD. mRNA vaccines can be rapidly designed, synthesized, and produced on a large scale in vitro, and they can induce effective protective immune responses, demonstrating the advantages of rapid development, easy preparation, and low biosafety risks. The design of untranslated regions is a key to enhancing the expression and efficacy of mRNA vaccines. In order to generate an efficient FMD mRNA vaccine, we designed three FMD P12A3C expression vectors with different untranslated regions and synthesized corresponding mRNAs. By comparing expression efficiency of these vectors and their mRNAs at different time points and in different cell lines, we found that the mRNA P12A3C-UTR3 had the best expression and universality. This study laid a foundation for the development of mRNA vaccines against FMD and provided a theoretical basis for the optimal sequence design of efficient mRNA.
Foot-and-Mouth Disease Virus/genetics* ; Animals ; RNA, Messenger/biosynthesis* ; Foot-and-Mouth Disease/immunology* ; Antigens, Viral/biosynthesis* ; Viral Vaccines/biosynthesis* ; Genetic Vectors/genetics* ; Cell Line ; Vaccines, DNA/immunology*

Bio-mineralization has emerged as a promising strategy to enhance vaccine immunogenicity. This study optimized the calcium phosphate (CaP) mineralization process of foot-and-mouth disease virus-like particles (FMD VLPs) to achieve high mineralization efficiency and scalability. Key parameters, including concentrations of Ca²⁺, HPO₄^2-, NaCl, and VLPs, as well as stirring speed, were systematically optimized. Stability of the scaled-up reaction system and immunogenicity of the mineralized vaccine were evaluated. Optimal conditions [25.50 mmol/L Ca(NO₃)₂, 15 mmol/L Na₂HPO₄, 300 mmol/L NaCl, 0.75 mg/mL VLPs, and 1 500 r/min] yielded CaP-mineralized VLPs (VLPs-CaP) with high mineralization efficiency, uniform morphology, and a favorable particle size. Scaling up the reaction by 25 folds maintained consistent mineralization efficiency and particle characteristics. Immunization in mice demonstrated that VLPs-CaP induced higher titers of specific antibodies and neutralizing antibodies than unmineralized VLPs (P < 0.05). Higher IgG2a/IgG1 ratio and enhanced IFN-γ secretion (P < 0.05) further indicated robust cellular immune responses. We establish a stable and scalable protocol for VLPs-CaP, providing a theoretical and technical foundation for developing high-efficacy VLPs-CaP vaccines.
Vaccines, Virus-Like Particle/immunology* ; Immunogenicity, Vaccine ; Calcium Phosphates/chemistry* ; Foot-and-Mouth Disease Virus ; Biomineralization ; Particle Size ; Animals ; Mice ; Antibodies, Neutralizing/blood* ; Antibodies, Viral/blood* ; Immunity, Cellular

Vaccination is a crucial strategy for the prevention and control of infectious diseases. Virus-like particles (VLPs), composed of structural proteins, have garnered significant attention as a novel type of vaccine due to their excellent safety and immunogenicity. However, similar to most vaccine antigens, VLPs exhibit insufficient thermal stability, which not only restricts the widespread application of vaccines but also increases the risk of vaccine inactivation. This study aims to enhance the stability and shelf life of VLPs derived from type A foot-and-mouth disease virus (FMDV) by employing vacuum freeze-drying technology. The optimal lyoprotectant formulation was determined through single-factor and combinatorial screening. Subsequently, the correlation between the immunogenicity of the freeze-dried vaccine and the content of FMDV VLPs was evaluated via a mouse model. The stability of FMDV VLPs before and after freeze-drying was further assessed by storing them at 4, 25, and 37 ℃ for varying time periods. Results indicated that the lyoprotectant formulation No.1, composed of 7.5% trehalose, 0.1% Tween 80, 50 mmol/L glycine, 1% sodium glutamate, and 3% polyvinylpyrrolidone (PVP), effectively preserved the content of FMDV VLPs during the vacuum freeze-drying process. The immunization trial in mice revealed that the levels of specific antibodies, immunoglobulin G1 (IgG1), interleukin-4 (IL-4), and neutralizing antibodies induced by freeze-dried FMDV VLPs were comparable to those induced by non-freeze-dried FMDV VLPs. The heat treatment results showed that the storage periods of freeze-dried FMDV VLPs at 4, 25, and 37 ℃ were significantly longer than those of non-freeze-dried FMDV VLPs. In conclusion, the selected lyoprotectant formulation effectively improved the stability of FMDV VLPs vaccines. This study provides valuable insights for enhancing the stability of novel subunit vaccines.
Freeze Drying/methods* ; Animals ; Foot-and-Mouth Disease Virus/immunology* ; Mice ; Vaccines, Virus-Like Particle/chemistry* ; Foot-and-Mouth Disease/immunology* ; Vacuum ; Drug Stability ; Mice, Inbred BALB C ; Viral Vaccines/immunology*

Lipid nanoparticles serve as a promising drug delivery system due to the good biocompatibility, non-immunogenicity, and high drug loading efficiency. However, unmodified lipid nanoparticles have limitations such as poor stability, easy hydrolysis, and rapid removal. To overcome these shortcomings, researchers have developed peptide modification, antibody modification, ligand modification, nucleic acid aptamer modification, and polysaccharide modification for lipid nanoparticles. Polysaccharides are a class of natural polymers, and the polysaccharide-modified lipid nanoparticles exhibit good biocompatibility, precise targeting, and low toxicity. Therefore, polysaccharide-modified lipid nanoparticles demonstrate great potential in clinical treatment. This review summarizes the preparation and application of polysaccharide-modified lipid nanoparticles, aiming to provide a reference for further research and development of new lipid nanoparticles.
Polysaccharides/chemistry* ; Nanoparticles/chemistry* ; Lipids/chemistry* ; Drug Delivery Systems ; Humans ; Drug Carriers/chemistry*

Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; Foot-and-Mouth Disease Virus

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA₂₅₇₋₂₆₄ peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLP(OVA)) in vitro after purification. The VLP(OVA) was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA₂₅₇₋₂₆₄ peptide into VP3 and its location. The results show that insertion of OVA₂₅₇₋₂₆₄ into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLP(OVA) in size was larger than VLPs, and the OVA₂₅₇₋₂₆₄ peptide was located on the surface of VLP(OVA).
Animals ; Escherichia coli ; genetics ; Foot-and-Mouth Disease ; virology ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Vaccines, Virus-Like Particle

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.
5' Untranslated Regions ; Capsid Proteins/metabolism* ; Foot-and-Mouth Disease Virus/physiology* ; Internal Ribosome Entry Sites ; Nucleic Acids/metabolism* ; Virus Assembly

AIM:To investigate the effect of liraglutide ( LG) on the expression of fibronectin type Ⅲdomain-containing protein 5 (FNDC5) in the C2C12 myotubes.METHODS:The C2C12 mouse myoblast cell line was induced to differentiation.Differentiated cells were stimulated with gradient concentrations (1 ~1000 nmol/L) of LG for different time (0 ~24 h).The effects of LG on the expression of FNDC5 and the activation of adenosine 5'-monophosphate ( AMP)-activated protein kinase ( AMPK) signaling pathway were determined .After pretreated with glucagon-like peptide-1 ( GLP-1 ) receptor antagonist exendin 9-39 , the inhibitor of Ca 2+/calmodulin-dependent protein kinase kinase 2 (CAMKK2), STO609, or the inhibitor of AMPK, Compound C, the LG-induced FNDC5 expression in C2C12 myotubes was examined.The expression of FNDC5 and the activation of AMPK were determined by Western blot .RESULTS: In C2C12 myotubes, LG promoted the expression of FNDC5 in a dose-and time-dependent manner .LG also activated AMPK signaling pathway .These effects of LG were partly abolished by exendin 9-39 , STO609 and Compound C .CONCLUSION:LG promotes the expression of FNDC5 via GLP-1 receptor in the C2C12 myotubes possibly through activation of the CAMKK2/AMPK signaling pathways .

Objective To develop a rapid and sensitive liquid chromatography-tandem mass spectrometric(LC-MS/MS) method for the determination of valproic acid in human plasma.Methods After treating human plasma sample by acetonitrile protein precipitation method,the analytes were separated on a Shimpack VP-ODS analytical column(150 mm×2.0 mm I.D,5 μm) with the mobile phase of methanol and 5 mmol/L ammonium acetate (55∶45,v/v)at a flow rate of 0.4 mL/min.Detection was carried out by adopting the multiple reaction monitoring(MRM) scanning mode in the API3200 triple quadrupole tandem mass spectrometer,electrospray ionization source,negative ion mode,selected monitoring ionic reactions were m/z 142.9→m/z 142.9(valproic acid) and m/z 179.0→m/z 179.0(1-sulfonic acid).Results Valproic acid and internal standard 1-sulfonic acid retention time were 3.03 min and 2.38 min respectively.The plasma valproic acid linear range was 0.800-80.0 μg/mL(r>0.99) with the lower limit of quantitation(LLOQ) 0.800 μg/mL.The intra-and inter-batch relative standard deviations(RSD) were both less than 15%,and the relative errors(RE) were within ±15%.The mean extraction recovery rate was(84.1±2.4)%,and the mean matrix effect factor was(104.3±2.0)%.In the stability study,valproic acid was found to be stable in plasma under various storage conditions.Conclusion This method is suitable for the determination of valproic acid in human plasma and human pharmacokinetic study of valproic acid semisodium sustained release tablet.

[ ABSTRACT] AIM:To investigate the role of hydrogen sulfide ( H2 S) on impaired wound healing in ob/ob mice and the underlying mechanism.METHODS:The ob/ob mice were randomly divided into 3 groups, including vehicle, in-sulin and NaHS for treatment.C57BL/6 mice were treated with vehicle as control.Full-thickness punch biopsy wounds were created on the mice.Firstly, H2 S concentrations in the skins and granulation tissues were measured.The mRNA ex-pression of cystathionineγ-lyase ( CSE) was detected by RT-qPCR.The protein expression of CSE and MMP-9 were deter-mined by Western blot.The neutrophil and monocyte/macrophage infiltration was analyzed by immunohistochemistry me-thod.The levels of tumor necrosis factor (TNF)-αand interleukin (IL)-6 were measured by ELISA.Collagen formation was measured by Masson staining.RESULTS:The H2 S levels in the skin and granulation were significantly decreased in ob/ob mice and increased in the NaHS-treated mice ( P<0.05 ) .CSE expression at mRNA and protein levels was significantly decreased in ob/ob mice compared with the control mice (P<0.05).The wound healing period was significantly shorter in NaHS group than that in vehicle-treated ob/ob mice group (P<0.05), in which the insulin group had no difference with vehicle ob/ob mice group.The neutrophil and monocyte/macrophage infiltration, and TNF-αand IL-6 levels were signifi-cantly increased in ob/ob groups, but were decreased in NaHS group ( P<0.01 or P<0.05 ) .Meanwhile, NaHS in-creased collagen formation in the granulation tissues of ob/ob mice.CONCLUSION:H2 S/CSE down-regulation contributes
to impaired wound healing in diabetes, which is alleviated by exogenous H2 S possibly through anti-inflammation.