Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Synergetic interaction of capsid proteins for virus-like particles assembly of foot-and-mouth disease virus (serotype O) from the inclusion bodies.

Recombinant virus-like particles (VLP) with single capsid protein have been successfully produced through prokaryotic system, but for those with multiple capsid proteins such as the foot-and-mouth disease virus (FMDV), this approach is more challenging. In this study, in vitro assembly of FMDV VLP was investigated with its capsids VP1, VP2 and VP3 separately expressed as inclusion bodies. After extraction and solubilization, three capsids were purified in denatured state through a flow-through model, increasing its purity to 90%. VLP assembly for FMDV was observed after diluting the mixture of denatured capsids in the ration of 1: 1: 1, while no VLP appeared if the separately diluted and refolded capsids were co-incubated. This result suggests certain synergetic interactions exist among the three capsids, which are crucial for FMDV VLP assembly. Sodium chloride and capsid protein concentration both greatly affect the assembling efficiency. After purification through size exclusion chromatography, VLP with similar diameter and morphology as inactivated FMDV were obtained, which elicited high IgG titers and B cell activation when vaccinated in mouse. It could also induce specific humoral and cellular immune responses in splenocytes proliferative experiments. Our study demonstrated the feasibility of in vitro assembling FMDV VLP from inclusion bodies of VP1, VP2 and VP3 for the first time.

Enhanced stability of foot-and-mouth disease vaccine antigens with a novel formulation.

The structural instability of inactivated Foot-and-mouth disease virus antigen hinders the development of vaccine industry. The use of an inexpensive, biocompatible formulation to slow down the degradation of antigen would address the problem. Here, phosphate-buffered saline (PBS) was showed to be effective in stabilizing 146S and hence determined as basic solution buffer. Excipients such as trehalose, sucrose, arginine, cysteine, calcium chloride, BSA and ascorbic acid were found to protect 146S from massive structural breakdown. Using orthogonal test, we confirmed the novel formulation as a combination of 5% (w/v) trehalose, 5% (w/v) sucrose, 0.05 M arginine, 0.01 M cysteine, 0.01 M calcium chloride, 1% (W/V) BSA and 0.001 M ascorbic acid in PBS. The formulation increased vaccine stabilization, with retention rate of 14% after storage at 4 °C for 14 months. Particle size for vaccine was at approximately 220 nm and physicochemical detecting findings were rarely abnormal in morphology and emulsion type. In summary, these results revealed that the novel formulation is beneficial to make the FMD vaccine more stable and effective, reducing the dependence on cold storage and delivery.

High level expression and purification of recombinant 3ABC non-structural protein of foot-and-mouth disease virus using SUMO fusion system.

The detection of antibody to non-structural protein (NSP) of Foot-and-mouth disease virus (FMDV) is the reliable diagnostic method for differentiating infected from vaccinated animals (DIVA). For this purpose, the detection of antibodies to non-structural 3ABC protein is suitable for identification of virus activity in the animals exposed to FMDV infection. However, large-scale production of recombinant 3ABC protein is challenging due to the formation of inclusion bodies in Escherichia coli and low yield due to protein aggregation during in vitro refolding. In this study, 3ABC gene was fused with SUMO (small ubiquitin-like modifiers) fusion system which significantly enhanced expression of recombinant 3ABC protein in E. coli. The solubility of the recombinant 6xHis-SUMO 3ABC fusion protein was improved by mild detergent treatment and purified through Ni-NTA chromatography under non-denaturing conditions which yielded 9 mg protein obtained from 1-L bacterial fermentation culture. The diagnostic potential of recombinant 3ABC protein was also tested by ELISA that provided reliable diagnostic performance (DSn = 92%, DSp = 94%) upon comparison with commercially available kit. The thermal stability of fusion protein was also tested which presented reliable performance at different temperatures. In conclusion, we presented SUMO fusion for the enhanced expression in E. coli and purification of active recombinant 3ABC protein using non-denaturing conditions without refolding steps. This protein can be used as a suitable diagnostic antigen to detect antibodies following FMDV infection.

Mooring Stone-Like Arg114 Pulls Diverse Bulged Peptides: First Insight into African Swine Fever Virus-Derived T Cell Epitopes Presented by Swine Major Histocompatibility Complex Class I.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently, no commercial vaccines are available. During the pig immune response, major histocompatibility complex class I (MHC-I) molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here, we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen 1*0101 (SLA-1*0101). We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cell receptor (TCR) docking. Thus, the solid Arg114 residue acts as a "mooring stone" and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cell recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC-I molecular anchor peptides and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen 1*0101 (SLA-1*0101), a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg114 of SLA-1*0101 as a "mooring stone" which pulls the peptide anchoring into the PBG in diverse "M"- or "n"-shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC-I and benefits the development of ASF vaccines.

Natural Phytochemicals, Luteolin and Isoginkgetin, Inhibit 3C Protease and Infection of FMDV, In Silico and In Vitro.

Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3Cpro) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3Cpro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 µM, respectively, whereas luteolin was less effective. Analyses of the protein-ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3Cpro. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization.

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the ßB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3E70 forms a key component of neutralizing epitopes, and the determination of the VP3E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

Effective Diagnosis of Foot-And-Mouth Disease Virus (FMDV) Serotypes O and A Based on Optical and Electrochemical Dual-Modal Detection.

Foot-and-mouth disease virus (FMDV) is a highly contagious disease that affects cloven-hoofed animals. The traditional diagnostic methods for FMDV have several drawbacks such as cross-reactivity, low sensitivity, and low selectivity. To overcome these drawbacks, we present an optical and electrochemical dual-modal approach for the specific detection of FMDV serotypes O and A by utilizing a magnetic nanoparticle labeling technique with resorufin ß-d-glucopyranoside (res-ß-glc) and ß-glucosidase (ß-glc), without the use of typical lateral flow assay or polymerase chain reaction. FMDV serotypes O and A were reacted with pan-FMDV antibodies that recognize all seven FMDV serotypes (O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3). The antigen-antibody complex was then immobilized on magnetic nanoparticles and reacted with ß-glc-conjugated FMDV type O or type A antibodies. Subsequently, the addition of res-ß-glc resulted in the release of fluorescent resorufin and glucose owing to catalytic hydrolysis by ß-glc. The detection limit of fluorescent signals using a fluorescence spectrophotometer was estimated to be log(6.7) and log(5.9) copies/mL for FMDV type O and A, respectively, while that of electrochemical signals using a glucometer was estimated to be log(6.9) and log(6.1) copies/mL for FMDV type O and A, respectively. Compared with a commercially available lateral flow assay diagnostic kit for immunochromatographic detection of FMDV type O and A, this dual-modal detection platform offers approximately four-fold greater sensitivity. This highly sensitive and accurate dual-modal detection method can be used for effective disease diagnosis and treatment, and will find application in the early-stage diagnosis of viral diseases and next-generation diagnostic platforms.

Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus.

An alternative vaccine design approach and diagnostic kits are highly required against the anticipated pandemicity caused by the South African Territories type 2 (SAT2) Foot and Mouth Disease Virus (FMDV). However, the distinct antigenicity and immunogenicity of VP1, VP0, and VP3 of FMDV serotype SAT2 are poorly understood. Similarly, the particular roles of the three structural proteins in novel vaccine design and development remain unexplained. We therefore constructed VP1, VP0, and VP3 encoding gene (SAT2:JX014256 strain) separately fused with His-SUMO (histidine-small ubiquitin-related modifier) inserted into pET-32a cassette to express the three recombinant proteins and separately evaluated their antigenicity and immunogenicity in mice. The fusion protein was successfully expressed and purified by the Ni-NTA resin chromatography. The level of serum antibody, spleen lymphocyte proliferation, and cytokines against the three distinct recombinant proteins were analyzed. Results showed that the anti-FMDV humoral response was triggered by these proteins, and the fusion proteins did enhance the splenocyte immune response in the separately immunized mice. We observed low variations among the three fusion proteins in terms of the antibody and cytokine production in mice. Hence, in this study, results demonstrated that the structural proteins of SAT2 FMDV could be used for the development of immunodiagnostic kits and subunit vaccine designs.

Predicting the Antigenic Relationship of Foot-and-Mouth Disease Virus for Vaccine Selection Through a Computational Model.

Foot-and-mouth disease virus (FMDV) is an antigenic-variable RNA virus that is responsible for the recurrence of foot-and-mouth disease in livestock and can be prevented and controlled using a vaccine with broad-spectrum protection. Current anti-genicity evaluation methods, which involve animal immunity experiments and serum preparation, are unable to fulfill the needs of high-throughput antigenicity measurements. This study designed an antigenicity scoring model to rapidly predict the antigenicity of FMDV. Antigenic-dominant sites were initially determined on the VP1 protein, a position-specific scoring matrix and physical chemical indexes were integrated to generate antigenicity descriptors. Independent tests showed a high accuracy of 0.848 and an AUC value of 0.889, indicating the good performance of the model in antigenicity measurement. When applying this model to historical data, annual antigenicity coverage of widely used vaccine strains was successfully evaluated, this was also supported by previous experiments. Furthermore, the utility of this model was extended to select potential broad-spectrum vaccines among 1,201 historical non-redundant strains to recommend potential univalent, bivalent and trivalent vaccine candidates. The results suggested that the computational model designed in this study could be used for the high-throughput antigenicity measurement of FMDV and could aid in vaccine development for preventing FMDV epidemics.

Mechanism of bio-macromolecule denaturation on solid-liquid surface of ion-exchange chromatographic media - A case study for inactivated foot-and-mouth disease virus.

Destruction of assembly structures has been identified as a major cause for activity loss of virus and virus-like particles during their chromatographic process. A deep insight into the denaturation process at the solid-liquid interfaces is important for rational design of purification. In this study, in-situ differential scanning calorimetry (DSC) was employed to study the dissociation process of inactivated foot-and-mouth disease virus (FMDV) during ion exchange chromatography (IEC) at different levels of pH. The intact FMDV known as 146S and the dissociation products were quantified by high performance size exclusion chromatography (HPSEC) and the thermo-stability of 146S on-column was monitored in-situ by DSC. Serious dissociation was found at pH 7.0 and pH 8.0, leading to low 146S recoveries of 12.3% and 43.7%, respectively. The elution profiles from IEC and HPSEC combined with the thermal transition temperatures of 146S dissociation (Tm1) from DSC suggested two denaturation mechanisms that the 146S dissociation occurred on-column after adsorption at pH 7.0 and during elution step at pH 8.0. By appending different excipients including sucrose, the improvement of 146S recovery and reduced dissociation was found highly correlated to increment of 146S stability on-column detected by DSC. The highest recovery of 99.9% and the highest Tm1 of 54.49 °C were obtained at pH 9.0 with 20% (w/v) sucrose. According to chromatographic behaviors and Tm1, three different dissociation processes in IEC were discussed. The study provides a perspective to understand the denaturation process of assemblies during chromatography, and also supplies a strategy to improve assembly recovery.

Negatively charged amino acids at the foot-and-mouth disease virus capsid reduce the virion-destabilizing effect of viral RNA at acidic pH.

Elucidation of the molecular basis of the stability of foot-and-mouth disease virus (FMDV) particles is relevant to understand key aspects of the virus cycle. Residue N17D in VP1, located at the capsid inner surface, modulates the resistance of FMDV virion to dissociation and inactivation at acidic pH. Here we have studied whether the virion-stabilizing effect of amino acid substitution VP1 N17D may be mediated by the alteration of electrostatic charge at this position and/or the presence of the viral RNA. Substitutions that either introduced a positive charge (R,K) or preserved neutrality (A) at position VP1 17 led to increased sensitivity of virions to inactivation at acidic pH, while replacement by negatively charged residues (D,E) increased the resistance of virions to acidic pH. The role in virion stability of viral RNA was addressed using FMDV empty capsids that have a virtually unchanged structure compared to the capsid in the RNA-filled virion, but that are considerably more resistant to acidic pH than WT virions, supporting a virion-destabilizing effect of the RNA. Remarkably, no differences were observed in the resistance to dissociation at acidic pH between the WT empty capsids and those harboring replacement N17D. Thus, the virion-destabilizing effect of viral RNA at acidic pH can be partially restored by introducing negatively charged residues at position VP1 N17.

Biomineralization improves the thermostability of foot-and-mouth disease virus-like particles and the protective immune response induced.

Virus-like particles (VLPs) are an ideal substitute for traditionally inactivated or attenuated viruses in vaccine production. However, given the properties of their native proteins, the thermal stability of VLPs is poor. In this study, calcium mineralization was used to fabricate foot-and-mouth disease virus (FMDV) VLPs as immunogenic core-shell particles with improved thermal stability. The biomineralized VLPs were stably stored at 24 °C and 37 °C for 13 and 11 days, respectively. Animal experiments showed that the biomineralized VLPs induced specific protective immunogenic effects, even after storage at 37 °C for 7 days. The biomineralized VLPs also effectively activated dendritic cells (DCs) to express high levels of surface MHC-II, costimulatory molecules, and proinflammatory cytokines. The DCs activated by the mineralized VLPs rapidly localized to the secondary lymphoid tissues and promoted the activation of the native T-cell population. These results suggest that the biomineralization of VLPs is an effective approach to vaccine production insofar as the mineralized shell provides an adjuvant effect which improves the immunogenicity of the VLPs. Biomineralization can also confer superior heat resistance on VLPs, an advantage in vaccine production. The successful development of thermally stable, biomineralized VLPs will reduce our dependence on cold storage and delivery.

Artificially designed hepatitis B virus core particles composed of multiple epitopes of type A and O foot-and-mouth disease virus as a bivalent vaccine candidate.

Recently, many countries, including China, have experienced a series of type A and O foot-and-mouth disease virus (FMDV) epidemics, causing serious economic losses. Although concerns about the safety of inactivated FMD vaccines have been raised, the development of a safe and effective subunit vaccine is necessary. We constructed two chimeric virus-like particles (VLPs; rHBc/AO and rHBc/AOT VLPs) displaying tandem repeats of B cell epitopes (VP1 residue 134-161 and 200-213) derived from type A and O FMDV and one T cell epitope (3 A residue 21-35) using the truncated hepatitis B virus core (HBc) carrier. Our results indicate that the chimeric HBc can self-assemble into VLPs with these FMDV epitopes displayed on the surface. Immunization with the chimeric VLPs induced specific IgG and neutralization antibodies against type A and O FMDV in mice. Compared with the commercial type A/O FMDV bivalent inactivated vaccine, rHBc/AO and rHBc/AOT VLPs significantly stimulated the production of Th1 type cytokines (IFN-γ and IL-2), whereas Th2 cytokine production (IL-4 and IL-10) was decreased. Compared with rHBc/AO, rHBc/AOT induced increased Th2 cytokine and specific IgG production. These results demonstrate that the VLPs constructed in the current study induced both humoral and cellular immune responses and may represent potential bivalent VLP vaccines targeting both FMDV type A and O strains.

Generation of acid resistant virus like particles of vaccine strains of foot-and-mouth disease virus (FMDV).

Foot-and-mouth disease (FMD) is a contagious viral disease affecting cloven hoofed livestock. Insect cell expressed virus like particles (VLPs) are potential alternative to overcome the limitations of inactivated vaccine. However, at pH < 6.5, virus particles disassociate into pentameric structure resulting in loss of antigenicity. Accordingly, we generated seven mutant VLPs containing mutations in the structural genes of FMDV vaccine strains (N17D and/or H145Y for serotypes O/IND/R2/75 and Asia1/IND/63/72; and H142D for serotype A/IND/40/00) by PCR based site directed mutagenesis. Acid resistant VLPs produced by baculovirus expression system were tested for acid stability at pH 7.5, 6.5, 6.0 and 5.5 followed by reactivity in sandwich-ELISA (s-ELISA), which revealed mutant-1 (N17D) of serotype O and Asia1 retained the antigenicity in s-ELISA even at pH 5.5 as compared to other VLPs and wild-types. Further, the 75S empty capsids obtained in sucrose density gradient, when tested in liquid phase blocking ELISA (LPBE) in comparison to cell culture antigen indicated that the VLPs were stable at acidic pH. Transmission electron microscopy of OM-1 confirmed the intact morphology of the empty VLPs. It is concluded that acid resistant VLPs could be useful for developing new generation vaccine or diagnostic for FMDV.

Determination of the optimal method for the concentration and purification of 146S particles for foot-and-mouth disease vaccine production.

After the severe outbreak of foot-and-mouth disease (FMD) in South Korea in 2010, the Korean government implemented a vaccination policy and set out to develop an FMD vaccine using a local FMD virus (FMDV) strain. As a part of the basic research for domestic FMD vaccine development, three methods commonly used for the concentration and purification of FMDV to produce FMD vaccine antigens were compared. Among common concentration methods, including polyethylene glycol (PEG) precipitation, ammonium sulfate precipitation, and ultrafiltration, the most effective method both for concentrating 146S particles and eliminating non-structural proteins (NSPs) was found to be PEG precipitation. Classical PEG precipitation showed the highest recovery of 146S particles (85.4%) with removing 99.8% of the other proteins, including NSPs. To the author's knowledge, this is the first study to compare the current three methods with regard to quantifying intact virus particles (146S). These findings may provide important insights for the development of new FMD vaccines using a local FMDV strain in the near future.

Deciphering foot-and-mouth disease (FMD) virus-host tropism.

The pattern of interactions between foot and mouth disease (FMD) viral protein 1 (VP1) with susceptible and resistant host integrins were deciphered. The putative effect of site-directed mutation on alteration of interaction is illustrated using predicted and validated 3D structures of VP1, mutated VP1 and integrins of Bos taurus, Gallus and Canis. Strong interactions were observed between FMDV-VP1 protein motifs at conserved tripeptide, Arg-Gly-Asp 143RGD145 and at domain 676SIPLQ680 in alpha-integrin of B. taurus. Notably, in-silico site-directed mutation in FMDV-VP1 protein led to complete loss of interaction between FMD-VP1 protein and B. taurus integrin, which confirmed the active role of arginine-glycine-aspartic acid (RGD) domain. Interestingly, in-vitro analysis demonstrates the persistence of the putative tropism site 'SIPLQ' in different cattle breeds undertaken. Thus, the attempt to decipher the tropism of FMDV at host receptor level interaction might be useful for future FMD control strategies through development of mimetic marker vaccines and/or host receptor manipulations. Communicated by Ramaswamy H. Sarma.

Development and modeling of two-dimensional fast protein liquid chromatography for producing nonstructural protein-free food-and-mouth diseases virus vaccine.

Concerns for the use of non-purified or incompletely purified inactivated foot-and-mouth disease (FMD) vaccine, like difficulties for differentiation vaccinated from infected animals, can be a motivation in order to develop methods based on size exclusion chromatography (SEC). In this study, a two dimensional size exclusion chromatography (2D-SEC) system was successfully constructed using two different SEC column media to achieve a high-throughput purification system for the cell culture-derived foot and mouth diseases virus (FMDV). A mathematical model was also utilized to predict and to get a better insight into the separation process. Column and the packing particles characteristics such as column void volume, total column volume, particle porosity and accessible particle porosity was acquired experimentally. Retention times and elution profile of two different molecules, blue dextran and bovine serum albumin, were used for evaluating the capability of SEC media for separating two critical impurities (residual DNA (rDNA) and non-structural protein (NSP)) from active ingredient of vaccine (FMDV particle). Experiments were carried out with two different commercial columns (XK 26/60) and (XK 16/100) and with four different packing media superdex 200 prep grade, sephacryl S-500 HR, Sephacryl S-400 HR and Sephacryl S-300HR. The mathematical model was first validated by experimental chromatographic data of different SEC media and was then used to propose the best 2D-SEC system for downstream processing of the FMDV vaccine. The loading capacity of the constructed 2D-SEC sample was increased to 12.5% of total column volume and the purity of the final product was more than 90%. The entire purification process was performed with 77% FMDV recovery and 79.1% virus yield. Based on the high-performance size exclusion chromatography (HPSEC), the purity of the final NSP-free FMDV was about 90% and over 94.6% of host cell DNA was removed. Analyses of the purified FMDV by HPSEC, transmission electron microscopy (TEM) and dynamic light scattering (DLS) indicated that the final product had spherical shape with mean size about 30 nm and their structure remained intact.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37 °C for 75 days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

[Using size-exclusion chromatography to quantify the 146S antigen in inactivated foot-and-mouth disease vaccine].

The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 µL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 µg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.