Scott R. Fry

The Diagnostic Sensitivity of Dengue Rapid Test Assays Is Significantly Enhanced by Using a Combined Antigen and Antibody Testing Approach

Background Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1–9 post-onset of illness but following seroconversion it can be difficult to detect in serum. Aims To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. Methodology The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. Key Results In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. Conclusions This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Structure aided design of chimeric antibiotics

The rise of antibiotic resistance is of great clinical concern. One approach to reducing the development of resistance is to co-administer two or more antibiotics with different modes of action. However, it can be difficult to control the distribution and pharmacokinetics of two drugs to ensure both concentrations remain within the range of therapeutic efficacy whilst avoiding adverse effects. Hybrid drugs, where two drugs are linked together with a flexible linker, have been explored, but the resultant large, flexible molecules can have poor bioavailability. We have developed a chimeric approach using click chemistry where the pharmacophores of two drugs are overlapped into a single smaller, more drug-like molecule. Design and selection of compounds were assisted by in silico structural docking. We prepared a series of compounds that include candidates showing activity against the targets of both trimethoprim; dihydrofolate reductase, and ciprofloxacin; DNA gyrase and topoisomerase IV. The resultant triazole containing molecules show modest, but broad spectrum activities against drug sensitive and resistant Gram-negative and Gram-positive bacteria, with no observable cytotoxicity.

Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay.

Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD= 1–0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model

ABSTRACT Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S–KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 103) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 102). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 104 and 2.6 × 104, respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

Development of homogeneous immunoassays based on protein fragment complementation

We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1kDa) to large (150kDa) analytes using TEM-1 beta-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Detection of HSV type-1 and type-2 IgG using an in vitro PCA based homogeneous immunoassay

Enzyme immunoassays (EIAs) are widely used in the clinical laboratory and research institutes for the detection of biologically relevant analytes. Almost all EIAs are heterogeneous in nature and require multiple steps of process. In contrast, homogeneous immunoassays (HA) offer a simplified one-step approach with a number of potential advantages over contemporary heterogeneous EIAs such as higher throughput and greater clinical utility. Utilizing TEM-1 beta-lactamase as a reporter enzyme, we have developed HAs based on in vitro protein fragment complementation (PCA) for the detection of antibodies and potentially be used for antigens or other biomarkers. In this proof-of-principle study we demonstrate the successful in vitro differentiation of anti-herpes simplex virus (HSV) type-1 and type-2 Immunoglobulin G (IgG) in human serum with high sensitivity and specificity.

Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. Here we present the structure of a cross-reactive, neutralizing antibody, 3E31, in complex with domain III (DIII) of the DENV envelope (E) protein and reveal a conserved, temperature-sensitive, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. We observed that 3E31 inhibits E-mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as-yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation. Finally, we show that, unlike cross-reactive fusion peptide-specific antibodies, 3E31 does not promote antibody-dependent enhancement of infection at sub-neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.