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*

The aim of this study was to compare the immune responses of C57BL/6 mice immunized with two pathogens, foot-and-mouth disease virus (FMDV) and Senecavirus A (SVA), and to provide clues for revealing the regulatory mechanisms of acquired immunity. Inactivated and purified FMDV and SVA antigens were used to immunize C57BL/6 mice respectively, and the mice immunized with PBS were taken as the control. The percentages of Th1 and Th2 cells in the spleen lymphocytes of mice in each group were analyzed by flow cytometry at 14 and 28 days after immunization. RNA-Seq was performed for the spleen. Mouse macrophages were stimulated with the antigens in vitro to examine the expression of the differentially expressed genes (DEGs) screened out. The results showed that 14 days after immunization, there was no significant difference in the magnitude of the Th1/Th2 immune response elicited by the FMDV and SVA antigens. After 28 days, the magnitudes of the Th1 and Th2 immune responses elicited by the SVA antigen were higher than those elicited by the FMDV antigen. RNA-Seq revealed two common DEGs, Rsad2 and Tspan8, between the two immunization groups, which indicated that the two genes may be involved in the activation of the Th1/Th2 immune responses by FMDV and SVA antigens. FMDV and SVA antigens stimulated macrophages to secrete interleukin (IL)-12 and IL-33 in vitro, and the expression of Tspan8 and Rsad2 was consistent with the RNA-Seq results. The expression of Rsad2 was regulated by type I interferons (IFNα, IFNβ). In this study, we obtained the DEGs involved in the immune responses to the two antigens in mouse spleen, which provides a molecular basis for investigating the immune response mechanisms induced by FMDV and SVA.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Mice ; Spleen/cytology* ; Mice, Inbred C57BL ; Antigens, Viral/genetics* ; Transcriptome ; Th1 Cells/immunology* ; Immunization ; Viral Vaccines/immunology* ; Th2 Cells/immunology* ; Foot-and-Mouth Disease/immunology* ; Interleukin-33/genetics* ; Female ; Macrophages/immunology* ; Picornaviridae

To reveal the innate immunity of mast cells against recombinant VP1-VP4 protein of foot-and-mouth disease virus (FMDV), mouse peritoneal mast cells (PMCs) were pulsed with recombinant VP1-VP4 protein. The supernatants harvested from PMCs cultures were applied to the high throughput ELISA array. Our results show that the expression levels of CCL19, L-selectin, CCL17, and TNF alpha released from PMCs pulsed with recombinant VP1-VP4 were significantly down-regulated compared with PMCs alone (P<0.001). Surprisingly, in comparison with PMCs alone, the expression levels of CCL19, IL-15, IL-9, G-CSF, and Galectin-1 in PMCs with the mannose receptor (MR) inhibitor were significantly up-regulated (Plt;0.01), and the expression level of IL-10 was also remarkably up-regulated (Plt;0.05). Importantly, the protein expression levels in PMCs treated with MR inhibitor were higher than PMCs pulsed with VP1-VP4, including IL-10, IL-17, CCL20, IL-15, IL-9, L-selectin, CCL17, TNF alpha, and CCL19 (Plt;0.01) as well as CCL21, and G-CSF (Plt;0.05). Differential expression analysis in bioinformatics shows that both L-selectin and CCL17 were recognized as differentially expressed protein molecules (Log2(ratio)≤-1) when compared with PMCs alone. Furthermore, the up-regulation of the expression levels of CCL20, CCL19, L-selectin, and IL-15 in PMCs treated with MR inhibitor was defined as differential expression (Log2(ratio)≥1). These data indicate that PMCs are capable of secreting CCL19, L-selectin, CCL17, and TNF alpha spontaneously and the recombinant VP1-VP4 has an inhibitive potential to PMCs during their performance of innate immune response. Given the protein expression levels from PMCs pre-treated with MR inhibitor were significantly increased, it can be deduced that immunosuppression of FMDV is presumably initiated by the VP1 recognition of MR on mast cells.
Animals ; Capsid Proteins ; immunology ; Cells, Cultured ; Cytokines ; immunology ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Interleukins ; immunology ; Mast Cells ; immunology ; Mice ; Proteome ; immunology ; Recombinant Proteins ; immunology ; Viral Structural Proteins ; immunology

OBJECTIVETo investigate the expression of vasoactive intestinal peptide (VIP) in peripheral blood of children with hand, foot and mouth disease and its significance.

METHODSAccording to the condition of the disease, 86 children with hand, foot and mouth disease were classified into phase 1 group (19 children) and phase 2 group (67 children). ELISA was used to measure the concentrations of plasma VIP, interferon-γ (IFN-γ), and interleukin-4 (IL-4) in peripheral blood. Flow cytometry was used to measure CD3, CD4, and CD8T lymphocyte subsets. RT-PCR was used for qualitative detection of enterovirus 71 (EV71) RNA in stool.

RESULTSCompared with the phase 1 group, the phase 2 group had a significantly higher positive rate of EV71-RNA (P<0.05) and significantly higher serum levels of IgG, IgA, IgM, and C3 (P<0.05). The phase 2 group had significantly lower proportions of peripheral CD3, CD4, and CD8T lymphocyte subsets than the phase 1 group (P<0.05), as well as significantly lower proportion of peripheral B cells and CD4/CD8ratio than the phase 1 group (P<0.05). The phase 2 group also had a significantly lower concentration of VIP in peripheral blood than the phase 1 group (P<0.05). In the 86 children with hand, foot and mouth disease, the concentration of VIP in peripheral blood was positively correlated with the proportion of CD4T lymphocyte subset and CD4/CD8ratio (r=0.533 and 0.532 respectively; P<0.05).

CONCLUSIONSVIP may be an important marker of the severity of hand, foot and mouth disease.

Biomarkers ; CD4-CD8 Ratio ; Child, Preschool ; Female ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Infant ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Male ; Severity of Illness Index ; Vasoactive Intestinal Peptide ; blood

OBJECTIVETo investigate the effect of continuous veno-venous hemofiltration (CVVH) on inflammatory mediators in children with severe hand, foot and mouth disease (HFMD), and to investigate its clinical efficacy.

METHODSA total of 36 children with stage IV HFMD were enrolled and randomly divided into conventional treatment group and CVVH group (n=18 each). The children in the CVVH group were given CVVH for 48 hours in addition to the conventional treatment. The levels of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and lactic acid in peripheral venous blood, heart rate, blood pressure, and left ventricular ejection fraction were measured before treatment and after 24 and 48 hours of treatment.

RESULTSAfter 24 hours of treatment, the conventional treatment group had a significantly reduced serum IL-2 level (P<0.01), and the CVVH treatment group had significantly reduced serum levels of IL-2, IL-6, IL-10, and TNF-α (P<0.05). After 48 hours of treatment, both groups had significantly reduced serum levels of IL-2, IL-6, IL-10, and TNF-α (P<0.01), and the CVVH group had significantly lower levels of these inflammatory factors than the conventional treatment group (P<0.01). After 48 hours of treatment, heart rate, systolic pressure, and blood lactic acid level were significantly reduced, and left ventricular ejection fraction was significantly increased in both groups, and the CVVH group had significantly greater changes in these indices except systolic pressure than the conventional treatment group (P<0.01).

CONCLUSIONSCVVH can effectively eliminate inflammatory factors, reduce heart rate and venous blood lactic acid, and improve heart function in children with severe HFMD.

Child, Preschool ; Cytokines ; blood ; Female ; Hand, Foot and Mouth Disease ; immunology ; physiopathology ; therapy ; Hemodynamics ; Hemofiltration ; Humans ; Infant ; Inflammation Mediators ; blood ; Male ; Ventricular Function, Left

Routine and emergency vaccination of small ruminants against foot-and-mouth disease (FMD) is mandatory in many endemic countries, yet data on the field effectiveness of the vaccines used is scarce. We conducted an investigation of a serotype O FMD outbreak that took place in a sheep and goat pen, and estimated the effectiveness of various routine vaccination statuses. We also evaluated the protection provided by colostrum administration and emergency vaccination. Animals which were routinely vaccinated twice were not clinically affected while disease incidence was observed among animals routinely vaccinated only once (p = 0.004 according to a two-sided Fisher's exact test). In groups vaccinated only once, there was a significant association between the average time that elapsed since last vaccination and the disease incidence (n = 5; Spearman correlation coefficient: r(s) = 1.0, p < 0.01). In addition, non-vaccinated lambs fed colostrum from dams vaccinated more than 2 months before parturition had a mortality rate of 33%. Administration of emergency vaccination 2 days after the occurrence of the index case was the probable reason for the rapid blocking of the FMD spread within 6 days from its onset in the pen.
Animals ; Colostrum ; Disease Outbreaks/veterinary ; Foot-and-Mouth Disease/*prevention & control ; Goat Diseases/*prevention & control ; Goats ; Immunization Schedule ; Sheep ; Sheep Diseases/*prevention & control ; Viral Vaccines/administration & dosage/*immunology

OBJECTIVETo clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.

METHODSVP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.

RESULTSVP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.

CONCLUSIONSVP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

Antibodies, Neutralizing ; blood ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Enterovirus A, Human ; genetics ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Immunoglobulin M ; blood ; Recombinant Proteins ; genetics ; immunology

Reverse-genetic engineering of foot and mouth disease virus (FMDV) can improve the productivity, antigen matching, antigen stability, immune response ability, and biological safety of vaccines, so vaccine candidates with anticipated biological characteristics can be promptly achieved. Negative influence in taming of virulent strains can also be decreased or avoided. Reverse genetics not only make up for deficiencies like limitation of viral nature, low success rate, and time and energy consuming, but also realize more active designing of vaccines. Therefore, reverse genetics is significant in improving integral quality and efficiency of vaccines. In this review, we use FMDV vaccines as an example to summarize improvement in biological characteristics of virulent strains and provide a reference for related researches.
Animals ; Antibodies, Viral ; immunology ; Foot-and-Mouth Disease ; immunology ; prevention & control ; virology ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Reverse Genetics ; Viral Vaccines ; genetics ; immunology