Avidity of Polyclonal Antibodies to Foot-and-Mouth Disease Virus in Bovine Serum Measured Using Bio-Layer Interferometry

Foot-and-mouth disease (FMD) is a disease of cloven-hoofed livestock caused by FMD virus (FMDV). FMD can be controlled through the use of inactivated vaccines, and it is well established that the protection afforded by FMD vaccines correlates strongly with neutralising antibody titres. However, the overall strength of binding, referred to as avidity, is also an important parameter with respect to the ability of antibodies to neutralise virus infection, and there is evidence that avidity can affect the level of protection afforded by FMDV vaccines. Here, as an alternative to modified enzyme-linked immunosorbent assays (avidity ELISAs) incorporating a chaotropic wash step, we used bio-layer interferometry (BLI) to measure the avidity of bovine polyclonal antibodies against FMDV capsids. We conducted preliminary experiments using recombinant FMDV capsids, as well as peptides representing antigenic loops, to demonstrate that the binding of monoclonal antibodies targeting specific antigenic sites could be detected using BLI. Subsequent experiments using polyclonal sera derived from FMD vaccinated cattle provided evidence of a positive correlation between the neutralising titre of the serum and the avidity as measured by BLI. Furthermore, we observed an increase in BLI avidity, as well as in the titre, in vaccinated animals upon challenge with the live virus.     

Development and laboratory validation of a lateral flow device for the detection of foot-and-mouth disease virus in clinical samples

A lateral flow device (LFD) for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV) was developed using a monoclonal antibody (Mab 1F10) shown to be pan-reactive to FMDV strains of each serotype by ELISA. The performance of the LFD was evaluated in the laboratory on suspensions of vesicular epithelia (304 positive and 1003 negative samples) from suspected cases of vesicular disease collected from 86 countries between 1965 and 2008 and negative samples collected from healthy animals. The diagnostic sensitivity of the LFD for FMDV was similar at 84% compared to 85% obtained by the reference method of antigen ELISA, and the diagnostic specificity of the LFD was approximately 99% compared to 99.9% for the ELISA. The device recognized FMDV strains of wide diversity of all seven serotypes but weaker reactions were often evident with those of type SAT 2, several viruses of which were not detected. Reactions with the viruses of swine vesicular disease and vesicular stomatitis that produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The test procedure was simple and rapid, and typically provided a result within 1-10min of sample addition. Simple homogenizers that could be used in field conditions for preparing epithelial suspensions were demonstrated to be effective for LFD application. These data illustrate the potential for the LFD to be used next to the animal in the pen-side diagnosis of FMD and for providing rapid and objective support to veterinarians in their clinical judgment of the disease.     

IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase

Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.     

Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia

Vascular endothelial growth factor (VEGF) is reported to play a neuroprotective role through a VEGF receptor, fetal liver kinase-1 (Flk-1) in vitro. We investigated whether reduction of Flk-1 could induce motor neuron loss in rat spinal cord by inhibiting the expression of Flk-1 in rat spinal cord using antisense oligodeoxynucleotides (ODNs) against the Flk-1 receptor. Rat spinal cord was repetitively exposed to 12% hypoxia, and the change of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway was examined. Intrathecal infusion of Flk-1 antisense ODNs for 7 days suppressed almost completely Flk-1 expression in the lumbar segment of the spinal cord and was followed by a hypoxic challenge with 12% oxygen for 1 h that was repeated for 7 more days. In the lumbar segment, we observed that reduced Flk-1 expression and hypoxic challenge for 7 days resulted in approximately 50% loss of motor neurons, in which the activation of Akt and ERK, that is, increased levels of phosphorylated-Akt and of phosphorylated-ERK by hypoxia, was markedly inhibited. In contrast, the reduction of Flk-1 expression alone did not induce motor neuron loss. These results suggest that VEGF exerts its protective effect on motor neurons against hypoxia-induced toxicity by the Flk-1 receptor through the PI3-K/Akt and the MEK/ERK signaling pathways.     

Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: a comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV (approximately 5 log) and VSV (approximately 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.     

Haemophilus influenzae porin induces Toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages

The production of proinflammatory cytokines is likely to play a major pathophysiological role in meningitis and other infections caused by Haemophilus influenzae type b (Hib). Previous studies have shown that Hib porin contributes to signaling of the inflammatory cascade. We examined here the role of Toll-like receptors (TLRs) and the TLR-associated adaptor protein MyD88 in Hib porin-induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Hib porin-induced TNF-alpha and IL-6 production was virtually eliminated in macrophages from TLR2- or MyD88-deficient mice. In contrast, macrophages from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, which are defective in TLR4 function, responded normally to Hib porin. Moreover anti-TLR2 antibodies but not anti-TLR4 antibodies significantly reduced Hib porin-stimulated TNF-alpha and IL-6 release from the human monocytic cell line THP-1. These data indicate that the TLR2/MyD88 pathway plays an essential role in Hib porin-mediated cytokine production. These findings may be useful in the development of alternative therapies aimed at reducing excessive inflammatory responses during Hib infections.     

Validation of a recombinant integrin αvβ6/monoclonal antibody based antigen ELISA for the diagnosis of foot-and-mouth disease

A sandwich ELISA using recombinant integrin αvβ6 as a capture ligand and serotype-specific monoclonal antibodies (Mabs) as detecting reagents has been compared with a polyclonal antibody based ELISA (using type-specific rabbit antibodies as capture and guinea pig antibodies as detectors), which is employed routinely at the FAO World Reference Laboratory for Foot-and-Mouth Disease (FMD), for the identification and serotyping of FMD virus (FMDV). The study used cell culture grown antigens (1351 FMDV positive) derived from suspected cases of vesicular disease collected from 86 countries between 1924 and 2011, those positive for the other vesicular diseases of swine vesicular disease (n = 25) and vesicular stomatitis (n = 45) and negative samples collected from uninfected cell cultures (n = 36). The diagnostic sensitivity of the assays was similar at 98.1% (polyclonal ELISA) compared to 97.9% (integrin/Mab ELISA) but the serotypic-specificity of the latter was vastly superior (96%) to that of the former (61.5%). Reactions with the viruses of swine vesicular disease and vesicular stomatitis, which produce clinically indistinguishable syndromes in pigs and cattle, did not occur. The integrin/Mab ELISA recognized FMDV strains of wide antigenic and molecular diversity of all seven serotypes and although some FMDV isolates were not detected, the greater specificity of the assay, while retaining test sensitivity comparable to the conventional assay, warrants its consideration for adoption for routine diagnostic use.