The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the β-ladder domain (amino acid residues 178–273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.