Foot-and-mouth Disease Virus Particles Research Articles

Promoting immunity with novel targeting antigen delivery vehicle based on bispecific nanobody.

As the most potent professional antigen presenting cells, dendritic cells (DCs) have been targeted in strategies to enhance vaccination efficacy. To date, targeted delivery has been mainly used for cancer therapy, with few studies focusing on vaccine antigens for animal epidemic diseases. In this study, we selected a series of mouse DC-specific nanobodies from a non-immunized camel. The four candidate nanobodies identified (Nb4, Nb13, Nb17, and Nb25), which showed efficient endocytosis of bone marrow-derived DCs, were evaluated as potential vaccine antigen targeted delivery vehicles. First, green fluorescent protein (GFP) was selected and four corresponding DCNb-GFP fusions were constructed for verification. Nb17-GFP was effective at promoting antibody production, inducing a cellular immune response, and increasing the IL-4 level. Second, foot-and-mouth disease virus (FMDV) and a FMDV-specific nanobody (Nb205) were selected and four bispecific nanobody DCNb-Nb205 fusions were generated to investigate the feasibility of a novel targeting antigen delivery vehicle. The resulting bispecific nanobody, Nb17-Nb205, could not only deliver FMDV particles instead of antigenic peptide, but also induced the production of specific antibodies, a cellular immune response, and IFN-γ and IL-4 levels upon immunization with a single subcutaneous injection. In conclusion, our results demonstrate the potential of bispecific nanobody as a novel and efficient DC-specific antigen delivery vehicle. This highlights the potential to expand targeted delivery to the field of animal epidemic diseases and provides a reference for the general application of nanotechnology in viral diseases.

Efficacy of Binary Ethylenimine in the Inactivation of Foot-and-Mouth Disease Virus for Vaccine Production in South Korea.

Foot-and-mouth disease (FMD) vaccines must be produced in a biosafety level 3 facility, so the FMD virus (FMDV) must be completely inactivated after amplification. The inactivation kinetics of FMDV during vaccine antigen production were assessed by evaluating whether the viral titer dropped below 10-7 TCID50/mL within 24 h of binary ethyleneimine (BEI) treatment. This study dealt with four FMD vaccine candidate strains for the efficacy of BEI treatment at different concentrations and temperatures to determine the optimal inactivation condition of each virus. Two domestic isolates, O/SKR/Boeun/2017 (O BE) and A/SKR/Yeoncheon/2017 (A YC), and two recombinant viruses, PAK/44/2008 (O PA-2) and A22/Iraq/24/64 (A22 IRQ), were investigated. The O BE and A22 IRQ required 2 mM BEI at 26 °C and 0.5 mM BEI at 37 °C for complete inactivation. The O PA-2 and A YC required 2 mM BEI at 26 °C and 1 mM BEI at 37 °C. Crucially, the yield of FMD virus particles (146S) in the viral infection supernatant was higher (>4.0 µg/mL) than those previously reported; additionally, there was little antigen loss, even after 24 h of treatment with 3 mM BEI. Overall, it is considered economical to produce FMD vaccines using these four kinds of viruses; therefore, these candidate strains will be prioritized for the manufacture of FMD vaccines in South Korea.

Size exclusion chromatography using large pore size media induces adverse conformational changes of inactivated foot-and-mouth disease virus particles

Size exclusion chromatography (SEC) of biomacromolecules using large pore size media and long column are usually necessary to obtain a satisfactory separation. However, the SEC separation of inactivated foot and mouth disease virus (FMDV) was found to induce some subtle but important conformational changes of FMDV in a pore-size and column length dependent manner. Here three Sephacryl media including S-300 HR, S-400 HR, and S-500 HR were tested, whose pore sizes were smaller than, similar to, and larger than the FMDV particles, respectively. High performance size exclusion chromatography (HPSEC) analyses showed that the FMDV after all these three SEC processes had earlier retention time, compared with that before SEC, but had no detectable difference in particle integrity. Longer SEC column led to more significant peak shifting in subsequent HPSEC analysis of FMDV. Further analyses indicated the SEC using larger pore size media induced more remarkable conformational changes and decrease in thermostability of FMDV, as well as decrease in immunogenicity in animal test. Fluorescence probe diffusion study suggested compared to SEC by S300, the compactness of the viral capsid after SEC by S400 and S500 was decreased, possibly due to more shear-induced FMDV particle rotation and inter-particle collision inside the media pores, as well as their interactions with the pore walls of the media during flowing through the column. Finally, a stabilization strategy by appending 5 mM CaCl2 in mobile phase of SEC separation was proposed and proved to efficiently maintain the conformation of the FMDV.

Comparison of High-Performance Liquid Chromatography with Sucrose Density Gradient Ultracentrifugation for the Quantification of Foot-and-Mouth Disease Vaccine Antigens.

Foot-and-mouth disease (FMD) causes substantial economic losses in the livestock industry. The protective immunizing component of the FMD virus (FMDV) is a ribonucleoprotein particle with a sedimentation coefficient of 146S. Size-exclusion high-performance liquid chromatography (SE-HPLC) was introduced to replace sucrose density gradient ultracentrifugation (SDG), which is the gold standard for the quantification of FMDV 146S particles. SE-HPLC showed a pattern similar to that of SDG; however, the two methods resulted in different quantities for the same amount of 146S particles. This study aimed to identify the reason for this disparity and adjust the difference between the two methods by employing a standard material. While SE-HPLC displayed all the virus particles in the peak fraction by SDS-PAGE and Western blotting, the virus particles were widely dispersed in multiple fractions, including peak fractions in the SDG. To adjust the difference between the two methods, a stable surrogate virus, bovine enterovirus, was devised to draw a standard curve, and the gap was reduced to <10%. To our knowledge, this is the first report to provide experimental evidence on the difference between SDG and SE-HPLC for the quantification of FMDV particles.

Determination of Appropriate BHK-21 Cell Line to Obtain High Infective Titer and 146S FMD Virus Particles

Foot and mouth disease (FMD) is one the most contagious diseases in cloven-hoofed animals and has a great potential for causing severe economic loss in susceptible animals. In the fight against FMD disease, inactive FMD vaccines are widely used. This study was carried out for the detection of suitable BHK-21 cell lines in order to get virus suspensions containing excess amounts of 146S FMD virus serotypes O, A and Asia-1, for FMD vaccine production. For this purpose, in addition to the BHK-21 An30 cell culture currently used in FMD vaccine production, BHK-21 An73 cell lines were passaged 20 times. In every 5 passages, infective titers and 146S virus particle quantities of FMD virus serotypes O, A and Asia-1 were measured. Additionally, karyotype analysis was performed on both cells at the 20th passage levels and the tests revealed that there was no significant change in the morphological features and chromosomal structures of the cells. It was determined that the infective titer and 146S virus particle quantities of serotypes O, A and Asia-1 of FMD virus were very high in the BHK-21 An73 cell line than they are available in the BHK-21 An30 cell line. The increases of 146S virus particle quantities of serotypes O, A and Asia-1 of FMD virus were statistically significant (p

Detection of Foot‐and‐Mouth Disease Virus Using a Polydiacetylene Immunosensor on Solid‐Liquid Phase

AbstractThe rapid detection of foot‐and‐mouth disease virus (FMDV) is vital for the prevention of foot‐and‐mouth disease outbreaks. In this study, a polyvinylidene difluoride (PVDF)‐supported polydiacetylene (PDA) immunosensor is developed to detect FMDV, in which a polyclonal antibody against the FMDV VP1 antigen is conjugated as a specific virus‐binding module without a linker. First, a liposome‐based immunosensor is generated for the FMDV VP1 protein in the form of photopolymerized PDA colloids. Then, the VP1‐specific PDA immunosensors are modified onto PVDF strip to enable the rapid and portable detection of FMDV. Detailed analyses are performed using ultraviolet‐visible spectroscopy, dynamic light scattering, transmission electron microscopy, and scanning electron microscopy. A blue‐to‐red color transition is observed in the presence of FMDV particles, indicating the potential applications of FMDV‐specific PDA immunosensors for use in solid‐phase detection as well as via liquid‐phase liposome platforms. Thus, this work provides a rapid and simple detection for FMDV.

Uncleaved 2A-peptide of foot-and-mouth disease virus can display foreign epitope-tag at the virion surface

Foot-and-mouth disease virus (FMDV) capsid precursor protein P1-2A is cleaved by viral-encoded 3C protease (3Cpro) to generate VP0, VP3, VP1 and 2A proteins. It was reported earlier that substitution of a single amino acid residue within the 2A peptide sequence (L2P) blocked the 3Cpro mediated VP1/2A cleavage and produced ‘self-tagged’ FMDV particles containing uncleaved 2A-peptide. To determine whether the uncleaved 2A-peptide can function as a target structure to harbour and display exogenous epitope on FMDV particles, a full-length FMDV cDNA clone containing a HA-tag within the uncleaved 2A-peptide sequence was constructed. Subsequently, chimeric marker FMDV, displaying a HA-tag on the viral surface was rescued through reverse genetics approach. The 2A-HA epitope tag-inserted recombinant chimeric FMDV serotype O was genetically stable through up to ten serial passages in cell culture and exhibited growth properties similar to the parental virus. Furthermore the surface displayed HA-epitope tag was able to react with anti-HA antibodies as determined by various immuno-assays. The results from our study suggest that the uncleaved 2A-peptide of FMDV is suitable to present foreign antigenic epitopes on the surface of FMD virion.

Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142→R or H142→F or H142→A substitutions resulted in non-infectious FMDV, H142→D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A.

Stabilizing effects of excipients on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles during storage as oil-emulsion vaccine

Most conventional foot-and-mouth disease virus (FMDV) vaccines contain oil-adjuvant. Their potency decreases upon prolonged storage. Intact (146S) FMDV particles can dissociate into 12S degradation products with a concomitant decrease in immunogenicity. We therefore measured virion stability in vaccines using two previously developed ELISAs to separately quantify 12S and 146S particles. Virions completely dissociated into 12S particles within 3 months after oil-emulsification. Dissociation occurred at a much lower rate in a comparable aqueous solution that was not oil-emulsified. Thus, oil-emulsification stimulates virion dissociation, presumably due to the protein denaturing effect of the oil–water interface. In real-time stability studies the stability of oil-adjuvanted virions of four different FMDV strains was significantly increased by addition of sucrose and BSA in a synergistic manner. Contrary to BSA addition, the effect of sucrose addition was concentration dependent. This study illustrates the importance of analysing antigen integrity after oil-emulsification and provides methods for FMDV vaccine stabilization.

Rescue of infective virus from a genome-length cDNA clone of the FMDV serotype O (IND-R2/75) vaccine strain and its characterization

We report here the construction and characterization of an infectious cDNA clone of the Indian vaccine strain of foot and mouth disease virus (FMDV) serotype O, IND-R2/75. Viral genome was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in five fragments and subsequently assembled sequentially in a plasmid vector to generate a complete cDNA clone, flanked by the T7 RNA polymerase promoter and poly (A) tail at 5′ and 3′ ends, respectively. Transfection of BHK-21 cells with the RNA transcribed from this genome-length cDNA construct allowed the recovery of infectious recombinant FMDV particles as evidenced by cytopathic effect in BHK-21 cells. Characterization of the recombinant virus revealed its similarity to the parental strain. Recombinant virus could be distinguished from the parental virus based on the presence of a unique marker sequence in the former, which was incorporated in the cDNA using a silent mutation. The virus showed no significant amino acid changes in the capsid-coding region when serially passaged up to ten times in BHK-21 cells, while retaining the marker sequence.

The effect of uniform capture molecule orientation on biosensor sensitivity: Dependence on analyte properties

Uniform orientation of capture molecules on biosensors has been reported to increase sensitivity. Here it is investigated which analyte properties contribute to sensitivity by orientation. Orientation of capture molecules on biosensors was investigated using variable domains of llama heavy-chain antibodies (VHHs) as capture molecule, and a surface plasmon resonance (SPR) chip as biosensor. Two VHHs were tested in this study: one recognizing foot-and-mouth disease virus (FMDV) and another recognizing the 16kDa heat-shock protein of Mycobacterium tuberculosis. SPR chips with randomly immobilized biotinylated VHHs were compared to streptavidin-coated SPR chips, on which similar quantities of oriented biotinylated VHHs were non-covalently immobilized. Analytes that differ in molecular weight, epitope number and epitope affinity were compared using the FMDV-recognizing VHH. When binding of intact FMDV particles (146S; 8200kDa) or pentameric FMDV coat protein aggregates (12S; 282kDa) was detected, a modest (1–2-fold) increase in sensitivity was observed. When a 26-residue peptide (3kDa) containing the epitope for VHH recognition was tested, much larger effects of capture molecule orientation (14-fold) on signal were observed. A 20–227-fold improvement was also observed when the epitope peptide was covalently linked to bovine serum albumin (67kDa) or R-phycoerythrin (240kDa). The results indicate that orientation of the capture molecule hardly affects high-affinity interactions, while it leads to strong improvements in sensitivity for lower-affinity interactions.

Inactivation of Nano- FMDV Type O/IRN/1/2007 Particles Infectivity Using Gamma Irradiation

Foot and Mouth Disease Virus (FMDV) is a vesicular and contagious disease of cloven-hoofed animals. This study is aimed to inactivate FMD Virus type O/2007/IRN particles by gamma irradiation with unaltered antigenicity. FMD Virus type O/2007/IRN was propagated on BHK21 cells. The virus titration was calculated by TCID 50 method. A 60 Co-gamma cell instrument Nordian, model 220 with dose rate 4.8 Gy/sec and activity of 20469 Ci was used for the inactivation of the frozen FMD virus samples. Safety test was done by cell culture method, as well as antigenicity of irradiated and non-irradiated virus samples were evaluated by Complement Fixation Test. Irradiated and non-irradiated FMDV particles were concentrated by centrifugation and studied under electron microscopy. The virus titration decreased gradually by increasing of gamma ray doses, according to the Dose/Survival curve for irradiated samples. The D 10 value factor (dose of gamma ray which can decrease 90% of virus population) and the optimum dose of gamma ray for FMDV type O/2007/IRN inactivation and unaltered antigenicity was obtained 5-5.5 kGy and 45-50 kGy, respectively. The irradiated inactivated FMD virus with unaltered antigenicity can be used as candidate radio-vaccine with superior safety.

Foot and mouth disease (FMD) virus: Quantification of whole virus particles during the vaccine manufacturing process by size exclusion chromatography

Foot and mouth disease (FMD) is a highly infectious viral disease that affects cattle, sheep, goats and swine causing severe economic losses worldwide. The efficacy of inactivated vaccines is critically dependent on the integrity of foot and mouth disease virus (FMDV) particles. The recommended method to quantify the active ingredient of vaccines is the 140S quantitative sucrose density gradient analysis. This method has been an immensely valuable tool over the past three decades but it is highly operator dependent and difficult to automate. We developed a method to quantify FMDV particles during the vaccine manufacturing process that is based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254nm. The method is linear in the 5–70μg/mL range, it is applicable to different FMDV strains, and has a good correlation with the 140S test. The proposed method uses standard chromatographic media and it is amenable to automation. The method has potential as a process analytical technology and for control of final product by manufacturers, international vaccine banks and regulatory agencies.

Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes

A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.

Characterization of foot-and-mouth disease virus antigen by surface-enhanced laser desorption ionization-time of flight-mass spectrometry in aqueous and oil-emulsion formulations

We have used a novel method, surface-enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS), to characterize foot-and-mouth disease virus (FMDV) vaccine antigens. Using specific capture with FMDV binding recombinant antibody fragments and tryptic digestion of FMDV antigens the spectral peaks representing the FMDV structural proteins VP1, VP2, VP3 and VP4 were identified. VP1 existed as 2 variants differing by 0.2 kDa and VP4 as 8 variants differing by 14–17 Da. Such heterogeneities have not been reported earlier. They could represent oxidation of VP4 and N-glycation of VP1. We also detected FMDV proteolysis upon incubation at elevated temperatures and impurities in FMDV antigen preparations. Finally, we could also characterize FMDV antigen present in emulsions with oil adjuvant by SELDI-TOF-MS. Such FMDV antigen retained the VP4 protein which is known to be specifically present in intact (146S) FMDV particles but absent from specific (12S) degradation products. This indicates that virions do not dissociate upon emulsification.

Biological Effect of Muller's Ratchet: Distant Capsid Site Can Affect Picornavirus Protein Processing

Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.

Construction of an infectious cDNA clone of foot-and-mouth disease virus type O1BFS 1860 and its use in the preparation of candidate vaccine

Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable,full-length cDNA clone of FMDV type O 1 BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEM R- - 7Zf(-). An 8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription.Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA,RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines.

Induction of a protective antibody response to FMDV in mice following oral immunization with transgenic Stylosanthes spp. as a feedstuff additive

The expression of antigens in transgenic plants has increasingly been used as an alternative to the classical methodologies for the development of experimental vaccines, and it remains one of the real challenges in this field to use transgenic plant-based vaccines effectively as feedstuff additives. We report herein the development of a new oral immunization system for foot and mouth disease with the structural protein VP1 of the foot and mouth disease virus (FMDV) produced in transgenic Stylosanthes guianensis cv. Reyan II. The transgenic plantlets were identified by polymerase chain reaction (PCR), Southern blotting, and northern blotting; and the production of VP1 protein in transgenic plants was confirmed and quantified by western blotting and enzyme-linked immunosorbent assays (ELISA). Six transformed lines were obtained, and the level of the expressed protein was 0.1-0.5% total soluble protein (TSP). Mice that were orally immunized using studded feedstuff mixed with desiccated powder of the transgenic plants developed a virus-specific immune response to the structural VP1 and intact FMDV particles. To our knowledge, this is the first report of transgenic plants expressing the antigen protein of FMDV as feedstuff additives that has demonstrated the induction of a protective systemic antibody response in animals. These results support the feasibility of producing edible vaccines from transgenic forage plants, and provide proof of the possibility of using plant-based vaccines as feedstuff additives.

Foot-and-Mouth Disease Virus Forms a Highly Stable, EDTA-Resistant Complex with Its Principal Receptor, Integrin αvβ6: Implications for Infectiousness

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin alphavbeta6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to alphavbeta6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin alphavbeta6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.

A segmented form of foot-and-mouth disease virus interferes with standard virus: A link between interference and competitive fitness

Serial passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells at high multiplicity of infection resulted in dominance of particles containing defective RNAs that were infectious by complementation in the absence of standard viral RNA. In the present study, we show that the defective FMDV particles interfere with replication of the cognate standard virus. Coinfections of defective FMDV with standard FMDV mutants that differ up to 151-fold in relative fitness have documented that the degree of interference is higher for low fitness than for high fitness standard virus. These comparisons suggest a likely overlap between those mechanisms of intracellular competition that underlie viral interference and those expressed as fitness differences between two viruses when they coinfect the same cells. Interference may contribute to the selective pressures that help maintain dominance of segmented defective RNAs over the standard FMDV genome.