Date Published: July 6, 2017
Publisher: Public Library of Science
Author(s): Kebaneilwe Lebani, Martina L. Jones, Daniel Watterson, Andrea Ranzoni, Renee J. Traves, Paul R. Young, Stephen M. Mahler, Daniel H. Libraty.
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.
Dengue virus infections are a significant public health burden. Dengue virus belongs to the genus flavivirus and is transmitted by the mosquito vectors Aedes aegypti and Aedes albopictus. Four distinct serotypes of DENV have been identified and often co-circulate together or with other flaviviruses such as Yellow Fever Virus, Japanese Encephalitis Virus, West Nile Virus and the emerging Zika virus (ZIKV) [1, 2]. 2010 population data and cartographic methods estimated the global dengue infection rate at 390 million infections annually, 96 million of which were symptomatic . Hyper-endemic areas in the tropics and sub-tropics particularly struggle with timely diagnosis of acute infection as well as epidemiological surveillance of the infective flavivirus and/or the infective dengue serotype.
Two phage libraries displaying scFv or Fab antibody fragments were interrogated for binders to DENV NS1. A subtractive biopanning strategy with three iterative rounds of biopanning for each DENV NS1 serotype and for each library was employed to isolate binders to immobilised, DENV rNS1. Binding to DENV rNS1 was confirmed by monoclonal phage ELISA, where 52 clones out of 90 screened from the DENV-1 pool were found to bind specifically to DENV-1 NS1. Similarly, for DENV-2, 8 out of 90; for DENV-3, 5 out of 90 and for DENV-4, 4 out of 90 were serotype specific. Other positive binders were also present but showed cross-reactivity with one or more other serotypes. After sequencing a selection of the positive clones, it was revealed that all of the DENV-1 clones were identical, all of the DENV-2 clones were identical and all of the DENV-3 clones were identical. There were two different sequences for the DENV-4 specific clones, but subsequent analysis showed that one of these clones was unable to bind natively sourced NS1. As such, four unique clones were taken forward for further characterisation: 9H2 (anti DENV-1), 4C11 (anti DENV-2), 7G11 (anti-DENV-3) and 6A5 (anti DENV-4).
The use of antibodies in diagnostic assays has progressively grown over the years and the formats in which the antibodies are used have become diverse. Commonly, ELISA is used where antibodies specific for the target antigen are used for capture and subsequent colorimetric detection of the antigen. Newer diagnostic platforms are now beginning to replace these traditional approaches and include formats such as lateral flow, biosensor and lab-on-a-chip approaches which can offer advantages for both assay cost, simplicity and time to readout. Despite these advantages, they all fundamentally depend on the availability of antibodies with high affinity and selectivity for their target antigen.