Eugene Boon Beng Ong

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

Phage display antibodies for diagnostic applications

With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.

Principles and application of antibody libraries for infectious diseases

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning.

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.Ts®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.Ts® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.

Cloning, expression, and purification of the hemolysin/cytolysin (HlyE antigen) from Salmonella enterica serovar Typhi: potential application for immunoassay development

The hemolysin (HlyE) protein of Salmonella enterica serovar Typhi was reported to be antigenic. This work describes the cloning, expression, and purification of a hexahistidine-tagged HlyE protein under native conditions. Immunoblot analysis and a competitive enzyme-linked immunosorbent assay using sera from typhoid patients showed the presence of HlyE-specific antibodies in circulation.

Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

Construction and characterization of a Salmonella enterica serovar Typhi tolC deletion mutant

Abstract Introduction Salmonella enterica serovar Typhi (S.Typhi), the organism responsible for typhoid fever, is a human specific pathogen that is able to survive in the macrophage and form biofilm in the host. TolC belongs to a family of outer membrane proteins that are found in all Gramnegative pathogenic bacteria. Together with two other proteins, AcrA and AcrB, TolC forms a channel through the outer membrane. Besides its role in the efflux of various molecules from the cell, TolC has been hypothesized to also play a key role in bacterial colonisation, adhesion and invasion of macrophages. Objectives To investigate the role of the outer membrane protein TolC of S.Typhi in macrophage infection and biofilm formation. Methods: The tolC deletion mutant (ΔTolC) strain of S.Typhi was constructed using a single-step gene knockout technique. S.Typhi cell invasion and biofilm formation ability of wild-type (WT) and ΔTolC strains were tested using an in vitro macrophage invasion assay and crystal violet biofilm assay, respectively. Results The ΔTolC strain was compromised in their ability to infect macrophages and promote biofilm formation. The ΔTolC strain invaded THP-1 macrophage cells very poorly compared with the WT (counts for wild-type were mean 5.1×105cfu/ml [n=4] compared with mean 1.6×105cfu/ml [n=4] for ΔTolC; p Conclusions Our data demonstrated the role of the S. Typhi TolC protein in biofilm formation and invasion of human macrophage cells.

Detection of Salivary IgA Antibodies Against the HlyE Antigen as a Diagnosis of Typhoid Fever.

INTRODUCTION The Salmonella typhi (S. typhi) haemolysin E protein (HlyE) has been shown to be a sensitive and specific antigen for the detection of typhoid fever through the detection of anti-HlyE antibodies in sera. Saliva can also be a useful diagnostic fluid as it also contains antibodies against bacterial pathogens. AIM This study aims to evaluate the potential detection of salivary anti-HlyE antibodies as a diagnosis of typhoid fever. MATERIALS AND METHODS Saliva was collected from acute typhoid patients (n=16) who presented at Hospital Universiti Sains Malaysia with prolonged fever of more than five days and were positive for S. Typhi blood culture. Saliva was also collected from convalescent typhoid patients (n=11), patients with other febrile fevers (n=15), and from healthy individuals (n=25). An ELISA was developed to detect the presence of IgA antibodies against HlyE in the saliva of typhoid patients. RESULTS The acute typhoid group had a higher mean absorbance value of 1.496 compared to the convalescent typhoid (0.538), other febrile fevers (0.678), and healthy individuals (0.457) group. CONCLUSION This study demonstrated the utility of salivary anti-HlyE IgA antibody as a biomarker for the diagnosis of typhoid fever. Follow-up studies with a larger sample size will allow the optimization of the sensitivity and specificity of the assay. This non-invasive method can be useful for mass screening programs.

In Silico Investigation of a HIV-1 Vpr Inhibitor Binding Site: Potential for Virtual Screening and anti-HIV Drug Design.

Present HIV antiviral therapy only targets structural proteins of HIV, but evidence shows that the targeting of accessory proteins will expand our options in combating HIV. HIV‐1 Vpr, a multifunctional accessory protein involved in viral infection, replication and pathogenesis, is a potential target. Previously, we have shown that phenyl coumarin compounds can inhibit the growth arrest activity of Vpr in host cells and predicted that the inhibitors’ binding site is a hydrophobic pocket on Vpr. To investigate our prediction of the inhibitors’ binding site, we docked the coumarin inhibitors into the predicted hydrophobic binding pocket on a built model of Vpr and observed a linear trend between their calculated binding energies and prior experimentally determined potencies. Subsequently, to analyze the inhibitor‐protein binding interactions in detail, we built homology models of Vpr mutants and performed docking studies on these models too. The results revealed that structural changes on the binding pocket that were caused by the mutations affected inhibitor binding. Overall, this study showed that the binding energies of the docked molecules are good indicators of the activity of the inhibitors. Thus, the model can be used in virtual screening to identify other Vpr inhibitors and for designing more potent inhibitors.

Overexpression, Purification and Validation of Antigenic Salmonella enterica Serovar Typhi Proteins Identified from LC-MS/MS

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.