Scientific Resources

BACKGROUND Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa,no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified,thus limiting development of diagnostic tests. METHODS Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively,in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein,calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves,associated with host IgG and IgM,were calculated to be 0.623 and 0.709 respectively,indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS While calflagin is conserved among different species of African trypanosomes,our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections. View Publication

BACKGROUND Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate,and is caused by the SFTS virus (SFTSV). SFTS is endemic to China,South Korea,and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas. METHODOLOGY/PRINCIPAL FINDINGS We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies,respectively. The Ag-capture system was capable of detecting at least 350-1220 TCID50/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (textgreater105 copies/ml) showed a positive reaction in the Ag-capture ELISA,whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (textless105 copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples,9 (75%) were positive for IgG antibodies against SFTSV. CONCLUSIONS The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia. View Publication

Although embryonal proteins have been used as tumor marker,most are not useful for detection of early malignancy. In the present study,we developed mouse monoclonal antibodies against fetal lung of miniature swine,and screened them to find an embryonal protein that is produced at the early stage of malignancy,focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2),an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies,with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover,tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS),inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues,eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung,similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma. View Publication

Summary The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14,when expressed in tumors,causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor,rather than host,is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia,thereby extending lifespan and improving quality of life for cancer patients. View Publication

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here,we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains,both lacking canonical export signals,are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs-named 'cyclonals'-effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation. View Publication

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore,this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile,indirect enzyme-linked immunosorbent assay (ELISA),an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated,and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate,CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay,3E7b blocks CHIKV attachment to permissive cells,possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection,3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively,these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design. View Publication

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1,ANXA11,CST4,PRDX5,PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1,ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. View Publication

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins,the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins,and as extension of previous work (Palomo et al.,2014),a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. View Publication

Helicobacter pylori causes cellular vacuolation in host cells,a cytotoxic event attributed to vacuolating cytotoxin (VacA) and the presence of permeant weak bases such as ammonia. We report here the role of γ-glutamyl transpeptidase (GGT),a constitutively expressed secretory enzyme of H. pylori,in potentiating VacA-dependent vacuolation formation in H. pylori-infected AGS and primary gastric cells. The enhancement is brought about by GGT hydrolysing glutamine present in the extracellular medium,thereby releasing ammonia which accentuates the VacA-induced vacuolation. The events of vacuolation in H. pylori wild type (WT)- and Δggt-infected AGS cells were first captured and visualized by real-time phase-contrast microscopy where WT was observed to induce more vacuoles than Δggt. By using semi-quantitative neutral red uptake assay,we next showed that Δggt induced significantly less vacuolation in AGS and primary gastric epithelial cells as compared to the parental strain (Ptextless0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably,vacuolation induced by WT was significantly reduced in the absence of GGT substrate,glutamine (Ptextless0.05) or in the presence of a competitive GGT inhibitor,serine-borate complex. Furthermore,the vacuolating ability of Δggt was markedly restored when co-incubated with purified recombinant GGT (rGGT),although rGGT itself did not induce vacuolation independently. Similarly,the addition of exogenous ammonium chloride as a source of ammonia also rescued the ability of Δggt to induce vacuolation. Additionally,we also show that monoclonal antibodies against GGT effectively inhibited GGT activity and successfully suppressed H. pylori-induced vacuolation. Collectively,our results clearly demonstrate that generation of ammonia by GGT through glutamine hydrolysis is responsible for enhancing VacA-dependent vacuolation. Our findings provide a new perspective on GGT as an important virulence factor and a promising target in the management of H. pylori-associated gastric diseases. View Publication

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably,several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP,but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site,revealing a mechanism of filovirus inhibition. View Publication

Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2,BMP-4,and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end,we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks,de novo bone formation was demonstrated by micro-CT imaging,histology,and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration,suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects,suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans,preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models. View Publication