Antibody discovery for development of a serotyping dengue virus NS1 capture assay

Abstract

Dengue virus (DENV) infections are a significant public health burden in tropical and sub-tropical regions of the world. Infections are caused by four different but antigenically related viruses which result in four DENV serotypes. The multifaceted nature of DENV pathogenesis hinders the sensitivity of assays designed for the diagnosis of infection. Different markers can be optimally detected at different stages of infection. Of particular clinical importance is the identification of acute viremia during the febrile phase of infection which is pivotal for management of infection. Non-structural protein 1 (NS1) has been identified as a good early surrogate marker of infection with possible applications in epidemiological surveillance and the development of blood screening assays. This contribution is towards using serotype-specificity to achieve specific and more sensitive diagnostic detection of DENV NS1. The general aim of this work is to isolate immune-reagents that can be used to develop an assay with improved sensitivity of DENV NS1 detection in a diagnostic setting. In this work, we sought to isolate serotype-specific antibodies that discern discreet antigenic differences in NS1 from each DENV serotype. Additionally, we also sought to isolate a pairing antibody that recognises NS1 from all four DENV serotypes (pan-reactive) for tandem capture of the DENV NS1. To achieve this, three naive, immunoglobulin gene libraries (a VH domain, a scFv and a Fab library) were interrogated for binders to recombinant NS1 antigen from all four DENV serotypes using phage display technology and various biopanning approaches. From biopanning experiments, four antibody fragments specific to NS1 from each DENV serotype as well as a panel of pan-reactive antibody fragments were isolated. The isolated antibody fragments were reformatted into fully assembled IgG1 antibodies for characterisation. In a sandwich ELISA, none of the pan-reactive antibodies were able to bind DENV NS1 in tandem with the serotype-specific antibodies. All four of the serotype-specific antibodies however, recognised their serotype-respective recombinant and native DENV NS1. The serotype-specific immunoglobulins showed equilibrium dissociation constants that ranged from µM to nM. Used as capture reagents in a sandwich ELISA with a commercial pan-reactive antibody as the detection probe, three of the antibodies (9H2 anti DENV-1 NS1, 7G11 anti DENV-3 NS1 and 6A5 anti DENV-4 NS1) were able to achieve clinically relevant limits of DENV NS1 detection. 4C11, an antibody against DENV-2 NS1 had the poorest performance and was subjected to affinity maturation through targeted mutagenesis of VH CDR3. A new variant A4, showed marginal 2-fold improvement in performance when incorporated into a limit of detection ELISA with the commercial pan-reactive antibody. Our data suggests that the four serotype-specific antibodies, including the new variant, would be useful for inclusion in a sandwich ELISA that uses NS1 to serotype DENV infections. To develop a complete DENV NS1 serotyping assay however, the serotype-specific antibodies will need to be used in a new iteration of a biopanning campaign to isolate an appropriate pan-reactive pairing partner