A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

Abstract

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Figure 1

Characterization of human mAbs to DENV. (a) Serotype specificity, assessed by ELISA, and reaction of 145 mAbs to DENV E protein, assessed by immunoblot analysis. (b) Serotype specificity of IB⁺ and IB⁻ mAbs. Data are representative of two experiments.

Figure 2

Epitope mapping of anti-DENV. (a) Epitope mapping with a panel of mutant VLPs (full results, Supplementary Table 2). Top, positions of substitutions in the domain structure of DENV E protein (horizontal stripe at top: DI, domain I; DII, domain II; DIII, domain III), including substitutions marking the fusion loop around Trp101 and disrupting the Asn153 glycosylation motif. Right and left margins, antibody groups (gray horizontal lines demarcate groups). (b) Positions of substitutions leading to >90% lower binding, mapped to either end of the E dimer (gray and orange circles). This model was based on the DENV-2 E dimer structure (PDB accession code 1OAN). (c) Three-dimensional reconstruction of the DENV-2 particle in complex with Fab 747(4)B7, calculated to a resolution beyond 10Å (Supplementary Fig. 3). The contour level shown corresponds to 2*sigma (the root mean square deviation of grid values in the map). The reconstruction is colored to provide radial depth (red (inner radii) to yellow green to cyan to blue (outer radii) (key)). Here, the projecting constant domain of the Fab is dark blue, the variable domain is cyan-green, the E protein shell is green-yellow and internal features (parts of the membrane) are seen in red through holes in the glycoprotein shell. Arrows point to the density of Fab 747(4)B7 bound to the same E dimer. (d) The density corresponding to Fab 747(4)B7 complex superimposed on the 3.5Å cryo-EM reconstruction of the DENV-2 virion, after subtraction of the E shell from the reconstruction of the complex. The three independent E and M proteins in the icosahedral asymmetric unit are in red for the subunit adjacent to the icosahedral twofold axes, are in yellow for that about the threefold axes, and are in cyan for that about the fivefold axis. Arrows point to the same density as in c. (e) A single E dimer showing Fab binding at the dimer interface matching the cluster of residues sensitive to binding, as determined by alanine scanning on DENV-2 VLPs in b.

Figure 3

mAbs to FLE versus mAbs to EDE in individual patients. Distribution of the responses to FLE and EDE (as percentages) by seven patients infected with DENV (top, patient identification numbers); numbers in centers indicate the number of antibodies from each patient. One copy of three duplicate antibodies (one EDE1 mAb and two EDE2 mAbs) from patient 752 with identical amino acid sequences was excluded from this and all other analyses.

Figure 4

Antibodies to EDE are potent and highly crossreactive in neutralization assays. Neutralization assays on Vero cells for nine representative mAbs (identifiers above plots) to FLE, EDE1 and EDE2 (top) of all four DENV serotypes (key) produced in C6/36 insect cells, presented as the 50% focus reduction neutralization titer (FRNT). Data are from two or three independent experiments, each with duplicate wells (mean ± s.e.m.).

Figure 5

EDE-specific antibodies have superior neutralizing activities. Neutralization of C6/36-DENV (a) or DC-DENV (b) by 138 mAbs (7 mAbs excluded because they did not react to DENV-2) at a final concentration of 0.05, 0.5 or 5 μg/ml (key (FRNT)), with Vero cells as the target. Below, classification of antibodies as FLE, EDE (five subgroups based on the results of VLP mapping: EDE1, EDE2, IB–3, IB–4 and IB–5) or non-FLE (IB⁺ antibodies that failed to map on the VLP); arrows indicate mAbs to EDE used elsewhere. Data represent two independent experiments each with duplicate wells.

Figure 6

Binding and neutralization of virus generated by insect cells and DCs. Titration curves for binding, measured by capture ELISA, and neutralization of DC-DENV and C6/36-DENV by three mAbs each from the FLE and EDE1 and EDE2 groups (presented as 50% FRNT values). AU, arbitrary units. Data are from two independent experiments representative of nine experiments each with anti-FLE and anti-EDE1 and seven experiments with anti-EDE2 (mean ± s.e.m.).

Figure 7

Binding of antibody to viral particles in various states of maturation. (a) ELISA of anti-prM and anti–E protein, to calculate a ratio of prM to E protein on viral particles from various cells (horizontal axis); results are presented relative to those of virus from LoVo cells, set as 100% (prM content). Each new batch of virus was tested, and the data were pooled (batches of viral preparations tested: 12 (C6/36), 21 (DC), 6 (293T), 5 (293T furin), 8 (LoVo) and 8 (Vero)). *P = 0.0084 and **P = 0.0005 (one-way analysis of variance (ANOVA), Kruskal-Wallis test). (b) Capture ELISA of the binding of mAbs to DENV-2 produced from C6/36, DC, 293T cells, furin-transfected 293T cells, LoVo cells or acid-treated DENV-2. Data are representative of two experiments with three mAbs to FLE, three EDE1 mAbs and three EDE2 mAbs, representative of eight mAbs to FLE, ten EDE1 mAbs and eight EDE2 mAbs (mean ± s.e.m.).

Figure 8

Reduced ADE with mAbs to EDE. (a) ADE assays on U937 cells infected with DENV-2 grown in either C6/36 cells or DCs, in the presence of titrations of mAbs to E protein that react to FLE or EDE; results are presented as median peak enhancement (fold). *P < 0.0001 (Mann-Whitney test). (b) NT₅₀ and NT₉₀ values for mAbs to FLE and EDE of C6/36-DENV and DC-DENV. Each symbol represents an individual mAb (black symbols, this study; gray symbols, from ref. : diamonds, mAb 752-2C8; triangles, mAb 753(3)C10; circles, mAb 747(4)A11; squares, 747(4)B7); small horizontal lines indicate the mean. Data are representative of two experiments.