Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the β-ladder domain

Abstract

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.

An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies

“…A dengue immune scFv-phage DHF.c library was constructed from convalescent sera of dengue patients as previously described . Additionally, a library DHF.a library was constructed from acute peripheral blood mononuclear cells (PBMCs) following the same protocol used for the DHF.c library, using the same set of patients.…”

“…A dengue immune scFv-phage DHF.c library was constructed from convalescent sera of dengue patients as previously described. 54 Additionally, a library DHF.a library was constructed from acute peripheral blood mononuclear cells (PBMCs) following the same protocol used for the DHF.c library, using the same set of patients. Twelve PBMCs were collected at the acute phase (day −1) from DENV infected patients in Khon Kaen and Songkhla hospitals during 2004−2009 and kept frozen.…”

Flaviviridae Nonstructural Proteins: The Role in Molecular Mechanisms of Triggering Inflammation

“…From the other side, some studies demonstrated that anti-DENV NS1 antibodies cross-react with clotting factors, platelets, and endothelial cells, leading to the production of proinflammatory cytokines and endothelial cell damage [156][157][158][159][160]; however, the contributions of these cross-reactive antibodies in DENV pathogenesis in vivo are still under debate. Taken together, the development of monoclonal Abs and vaccines against DENV based on NS1 should be carefully performed, and the epitopes of NS1 able to elicit auto-antibodies in the organisms should be omitted [161,162].…”