Abstract
The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.
Highlights
Dengue virus infections are a significant public health burden
Our aim was to isolate antibodies which could discern between the four serotypes of dengue virus (DENV), which could potentially be used to improve the utility of DENV Nonstructural protein 1 (NS1) diagnostic Enzyme Linked Immunosorbent Assay (ELISA), by adding a serotyping capability
A subtractive biopanning strategy with three iterative rounds of biopanning for Serotype specific dengue virus NS1 antibodies isolated by phage display each DENV NS1 serotype and for each library was employed to isolate binders to immobilised, DENV Recombinant NS1 (rNS1)
Summary
Dengue virus belongs to the genus flavivirus and is transmitted by the mosquito vectors Aedes aegypti and Aedes albopictus. Hyper-endemic areas in the tropics and sub-tropics struggle with timely diagnosis of acute infection as well as epidemiological surveillance of the infective flavivirus and/or the infective dengue serotype. Diagnosis of dengue infections can be achieved by several means, including virus culture, nucleic acid tests, IgG and IgM serology tests and antigen detection tests such as those that detect NS1 [5]. Each of these tests has its advantages and limitations which have been well reviewed [6, 7]
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