Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV), which seriously endangers the development of animal husbandry. The inactivated FMD vaccine is the main product for the prevention and control of FMD, which has been successfully applied to control the pandemic and outbreak of FMD. However, the inactivated FMD vaccine also has problems, such as the instability of antigen, the risk of spread of the virus due to incomplete inactivation during vaccine production, and the high cost of production. Compared with traditional microbial and animal bioreactors, production of antigens in plants through transgenic technology has some advantages including low cost, safety, convenience, and easy storage and transportation. Moreover, since antigens produced from plants can be directly used as edible vaccines, no complex processes of protein extraction and purification are required. But, there are some problems for the production of antigens in plants, which include low expression level and poor controllability. Thus, expressing the antigens of FMDV in plants may be an alternative mean for production of FMD vaccine, which has certain advantages but still need to be continuously optimized. Here we review the main strategies for expressing active proteins in plants, as well as the research progress on the expression of FMDV antigens in plants. We also discuss the current problems and challenges encountered, with the aim to facilitate related research.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Foot-and-Mouth Disease/prevention & control* ; Antigens, Viral/genetics* ; Viral Vaccines

To further enhance the immune effect of the foot-and-mouth disease (FMD) virus-like particles (VLPs) vaccine, this study prepared FMDV VLPs-zeolitic imidazolate (framework-8, ZIF-8) complexes with different particle sizes. We used a biomimetic mineralization method with Zn²⁺ and 2-methylimidazole in different concentration ratios to investigate the effect of size on the immunization effect. The results showed that FMDV VLPs-ZIF-8 with three different sizes were successfully prepared, with an approximate size of 70 nm, 100 nm, and 1 000 nm, respectively. Cytotoxicity and animal toxicity tests showed that all three complexes exhibited excellent biological safety. Immunization tests in mice showed that all three complexes enhanced the titers of neutralizing and specific antibodies, and their immune effects improved as the size of the complexes decreased. This study showed that ZIF-8 encapsulation of FMDV VLPs significantly enhanced their immunogenic effect in a size-dependent manner.
Animals ; Mice ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Antibodies, Neutralizing ; Immunity, Humoral ; Immunization ; Vaccines, Virus-Like Particle ; Antibodies, Viral ; Viral Vaccines

Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Capsid Proteins ; Viral Proteins/metabolism* ; Foot-and-Mouth Disease/prevention & control* ; Tetracyclines/metabolism* ; Viral Vaccines ; Antibodies, Viral ; Mammals/metabolism*

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*

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

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.
Amino Acids ; Animals ; Capsid Proteins/genetics* ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; Kinetics ; Vaccines, Virus-Like Particle/genetics* ; Viral Vaccines/genetics*

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

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.
Animals ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Viral Nonstructural Proteins/genetics* ; Viral Vaccines

Foot-and-mouth disease (FMD) is one of the most important livestock diseases in East Africa with outbreaks reported annually that cause severe economic losses. It is possible to control disease using vaccination, but antigenic matching of the vaccine to circulating strains is critical. To determine the relationship between foot-and-mouth disease viruses circulating in districts along the Uganda and Tanzanian border between 2016 and 2017 and currently used vaccines, phylogenetic analysis of the full VP1 virus sequences was carried out on samples collected from both sides of the border. A total of 43 clinical samples were collected from animals exhibiting signs of FMD and VP1 sequences generated from 11 of them. Eight out of the 11 sequences obtained belonged to serotype O and three belonged to serotype A. The serotype O sequences obtained showed limited nucleotide divergence (average of 4.9%) and belonged to topotype East Africa-2, whereas the most common O-type vaccine strain used in the region (O/KEN/77/78) belonged to East Africa-1. The serotype A viruses belonged to topotype Africa-G1 (average nucleotide divergence 7.4%), as did vaccine strain K5/1980. However, vaccine strain K35/1980 belonged to Africa G VII with an average sequence divergence of 20.5% from the study sequences. The genetic distances between current vaccine strains and circulating field strains underscores the crucial need for regular vaccine matching and the importance of collaborative efforts for better control of FMD along this border area.
Africa ; Africa, Eastern ; Animals ; Disease Outbreaks ; Foot-and-Mouth Disease Virus ; Foot-and-Mouth Disease ; Livestock ; Serogroup ; Tanzania ; Uganda ; Vaccination ; Vaccines