Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay

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.

Development of Dengue Virus Serotype–Specific NS1 Capture Assays for the Rapid and Highly Sensitive Identification of the Infecting Serotype in Human Sera

“…Due to the significant amino acid sequence homology of DENV serotypes (5,6), the generation of mAbs exclusively recognizing only one of the four variants is challenging. Recently, the isolation of anti-NS1 Abs by a combination of phage display and a subtractive biopanning strategy to direct Ab selection toward serotype-specific epitopes has been reported (26). A disadvantage of this approach is that Abs lack the in vivo process of affinity maturation that occurs in natural humoral immune responses.…”

Serotype-specific detection of dengue viruses in a nonstructural protein 1-based enzyme-linked immunosorbent assay validated with a multi-national cohort

“…Lebani et al utilized phage display to isolate serotype-specific NS1 DENV antibodies for an ELISA. Along with using costly machinery to detect fluorescent microsphere beads in their assay, they used human serum spiked with virus as opposed to infected patient samples in their assays [ 34 ]. Röltgen and Lai developed a serotype NS1 DENV ELISA but they each used approximately 60 positive serum samples, of which all or a majority originated from Asia [ 27 , 28 ].…”

“…assay, they used human serum spiked with virus as opposed to infected patient samples in their assays [34]. Röltgen and Lai developed a serotype NS1 DENV ELISA but they each used approximately 60 positive serum samples, of which all or a majority originated from Asia [27,28].…”

Phage Display as a Strategy to Obtain Anti-flavivirus Monoclonal Antibodies

“…To characterize the diagnostic potential of the antibodies, MAbs were addressed [71]. Lebani et al [72] isolate four serotype-specific human antibodies through a negative selection strategy. Each MAb was specific for NS1 from a DENV serotype, without cross-linking.…”