One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection, Antigen Preparation, and Epitope Mapping.

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His6-tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format.

Immunoassay-Compatible Inactivation of SARS-CoV-2 in Plasma Samples for Enhanced Handling Safety.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri-n-butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings.

Extracellular galectin-3 counteracts adhesion and exhibits chemoattraction in Helicobacter pylori-infected gastric cancer cells.

Galectin-3 (Gal-3) is a ß-galactoside lectin that is upregulated and rapidly secreted by gastric epithelial cells in response to Helicobacter pylori infection. An earlier study reported the involvement of H. pylori cytotoxin-associated gene A (cagA) in the expression of intracellular Gal-3. However, the role of extracellular Gal-3 and its functional significance in H. pylori-infected cells remains uncharacterized. Data presented here demonstrate secretion of Gal-3 is an initial host response event in gastric epithelial cells during H. pylori infection and is independent of CagA. Previously, Gal-3 was shown to bind to H. pylori LPS. The present study elaborates the significance of this binding, as extracellular recombinant Gal-3 (rGal-3) was shown to inhibit the adhesion of H. pylori to the gastric epithelial cells. Interestingly, a decrease in H. pylori adhesion to host cells also resulted in a decrease in apoptosis. Furthermore, the study also demonstrated a chemoattractant role of extracellular rGal-3 in the recruitment of THP-1 monocytes. This study outlines the previously unidentified roles of extracellular Gal-3 where it acts as a negative regulator of H. pylori adhesion and apoptosis in gastric epithelial cells, and as a chemoattractant to THP-1 monocytes. Our findings could contribute to the better understanding of how Gal-3 acts as a modulator under H. pylori-induced pathological conditions.

Instrumental Role of Helicobacter pylori γ-Glutamyl Transpeptidase in VacA-Dependent Vacuolation in Gastric Epithelial Cells.

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 (P<0.05) indicating that GGT potentiates the vacuolating effect of VacA. Notably, vacuolation induced by WT was significantly reduced in the absence of GGT substrate, glutamine (P<0.05) 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.

Helicobacter pylori γ-glutamyl transpeptidase: a formidable virulence factor.

Helicobacter pylori (H. pylori) produce an enzyme known as γ-glutamyl transpeptidase (HpGGT) that is highly conserved and common to all strains. HpGGT has been gaining increasing attention as an important virulence factor of the bacterium, having been demonstrated to be an important colonization factor in several animal models and has also recently been strongly associated with the development of peptic ulcer disease. From the results of various independent researcher groups, it is clear that HpGGT acts through several pathways to damage gastric epithelial cells including the induction of apoptosis and cell cycle arrest, production of reactive oxygen species leading to DNA damage, promotion of inflammation by increasing cyclooxygenase-2 and interleukin-8 expression, and upregulation of heparin-binding epidermal growth factor-like growth factor resulting in cell survival and proliferation. In addition, the potential role of HpGGT in promoting gastric carcinogenesis will also be discussed in this review. Apart from affecting the gastric epithelium, HpGGT also has immunomodulatory actions on host immune cells where it displays an antiproliferative effect on T cells by inducing cell cycle arrest and also works with other H. pylori virulence factors to skew dendritic cells towards a tolerogenic phenotype, possibly contributing to the persistence of the pathogen in the gastric mucosa.

Helicobacter pylori gamma-glutamyl transpeptidase is a pathogenic factor in the development of peptic ulcer disease.

BACKGROUND & AIMS: gamma-Glutamyl transpeptidase (GGT) has been reported to be a virulence factor of Helicobacter pylori associated with bacterial colonization and cell apoptosis. But its mechanism of pathogenesis is not firmly established. This study aims to examine its role in H pylori-mediated infection. METHODS: Various H pylori isogenic mutants were constructed by a polymerase chain reaction (PCR) approach. H pylori native GGT protein (HP-nGGT) was purified with ion-exchange and gel-filtration chromatography. Generation of H2O2 was measured with fluorimetric analysis, whereas nuclear factor-kappaB (NF-kappaB) activation was determined by luciferase assay and Western blot. Cytokine production was examined by enzyme-linked immunoabsorbent assay and real-time PCR. DNA damage was assessed with comet assay and flow cytometry. The GGT activity of 98 H pylori isolates was analyzed by an enzymatic assay. RESULTS: Purified HP-nGGT generated H2O2 in primary gastric epithelial cells and AGS gastric cancer cells, resulting in the activation of NF-kappaB and up-regulation of interleukin-8 (IL-8) production. In addition, HP-nGGT caused an increase in the level of 8-OH-dG, indicative of oxidative DNA damage. In contrast, Deltaggt showed significantly reduced levels of H2O2 generation, IL-8 production, and DNA damage in cells compared with the wild type (P<.05). The clinical importance of GGT was indicated by significantly higher (P<.001) activity in H pylori isolates obtained from patients with peptic ulcer disease (n=54) than isolates from patients with nonulcer dyspepsia (n=44). CONCLUSION: Our findings provide evidence that GGT is a pathogenic factor associated with H pylori-induced peptic ulcer disease.