Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Abstract

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To delineate bNAb targets, we characterized 28 monoclonal antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies revealed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against flaviviruses with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested a memory origin and divergent somatic hypermutation pathways for these bNAbs, and a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a novel epitope that can be exploited for vaccine design. E monomeric subunits within the dimer. A subset of these E dimer epitope (EDE)-specific bNAbs potently neutralize not only DENV1-4, but also ZIKV, owing to the high conservation of the EDE, which overlaps the prM binding site on E ( Barba-Spaeth et al., 2016;Dejnirattisai et al., 2015;Rouvinski et al., 2015). The exciting discovery of the EDE class of bNAbs highlights the potential for an epitope-focused flavivirus vaccine strategy.As multiple specificities are likely required to provide maximum coverage of diverse circulating viral variants (Bell et al., 2019;Doria-Rose et al., 2012;Goo, Jalalian-Lechak, Richardson, & Overbaugh, 2012;Katzelnick et al., 2015; Keeffe et al., 2018; Kong et al., 2015), in this study, we aimed to define novel sites on the flavivirus E protein that can be targeted by bNAbs. By characterizing 28 monoclonal antibodies from the plasmablasts of two DENV-infected individuals, we identified J8 and J9, clonally related bNAbs that neutralized DENV1-4 in the low picomolar range. The major recognition determinants for J8 and J9 were in E protein DI, distinct from previously characterized bNAbs. Analysis of the corresponding B cell repertoire revealed divergent evolution of J8 and J9, suggesting multiple evolutionary pathways to generate bNAbs within this lineage. Our work identifies both viral and host determinants of the development of DENV bNAbs that can guide immunogen design and evaluation. Results Identification of cross-reactive neutralizing antibodies from clonally expanded plasmablasts of DENV-infected individualsWe previously profiled the single-cell transcriptomics of peripheral blood mononuclear cells (PBMCs) from six dengue patients and four healthy individuals (Zanini et al., 2018). In two DENV-infected patients (013 and 020), we identified 15 clonal families comprising a total of 38 unique paired heavy (VH) and light (VL) chain IgG1 plasmablast sequences, some of which were hypermutated (1.67% to 10.77% for VH, 0.67% to 7.22% for...

Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection

“…To rule out a mouse antibody-specific artifact, we next performed the above ADE assays using a broadly reactive human monoclonal anti-DENV IgG antibody, J9 [ 75 ]. In these assays, TBC1D24 KO ( Figure 2A ) and SV2B KO ( Figure 2B ) each resulted in a ∼50% reduction in AUC compared to WT cells.…”

“…TBC1D24 and SV2B are required for efficient ADE but not direct infection. ( A-B ) (Left) Representative dose-response ADE curves for K562 ( A ) TBC1D24 KO clone ( B ) SV2B KO clone, and genetically trans -complemented K562 KO cells infected with DENV2-GFP in the presence of serially diluted human anti-DENV IgG monoclonal antibody J9 [ 75 ]. In each experiment, K562 WT cell pool and a FcgRIIa KO clone was included as a control.…”

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies

“…Previous efforts to characterize antigenicity of flavivirus surface proteins have not been able to distinguish between functionally required residues for neutralization versus binding [23][24][25][26]28 . Other efforts, such as phage display platforms to perform alanine scanning of dengue virus and small scale site-directed mutagenesis could only identify linear antibody epitopes, and only provide limited information on a few domains of E 16,17,38 . In contrast, our approach comprehensively measures the effect of all single amino acid mutations on neutralization by antibodies with complex quaternary epitopes.…”

The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies

“…Previous efforts to characterize antigenicity of flavivirus surface proteins have not been able to distinguish between functionally required residues for neutralization versus binding ( 23 – 26 , 28 ). Other efforts, such as phage display platforms to perform alanine scanning of dengue virus and small scale site-directed mutagenesis, could only identify linear antibody epitopes and only provide limited information on a few domains of E ( 16 , 17 , 39 ). In contrast, our approach comprehensively measures the effect of all single amino acid mutations on neutralization by antibodies with complex quaternary epitopes.…”

“…Thus, there is immense interest in both developing vaccines that elicit broad and potent neutralizing antibody responses, as well as isolating and leveraging broadly neutralizing antibodies as biological therapeutics. Toward this end, investigations have examined human antibody repertoires and isolated a select few antibodies that can broadly and potently neutralize Zika virus and all serotypes of dengue virus ( 16 – 20 ). These antibodies all target envelope (E) protein, which is the major antigenic target in both Zika virus and dengue virus ( 21 , 22 ).…”