Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop

Abstract

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

Fig 1

Broadly reactive patient-derived monoclonal antibodies. (A) DENV-1, -2, -3, and -4 glycosylated antigens were captured on ConA-coated plates and probed with dilutions of patient 8C and DA003 sera. The data points show the means of one experiment with three replicates. The error bars show standard deviations. (B) DENV-1, -2, -3, and -4 glycosylated antigens were captured on ConA-coated plates and probed with dilutions of hMAbs D11C and 1.6D. Representative data show the means of one experiment with three replicates. The error bars show standard deviations. (C) LLC-MK₂ cells infected with DENV-1, -2, -3, and -4 at an MOI of 0.002 were probed with hMAbs 4.8A, D11C, and 1.6D and imaged by confocal microscopy. The nuclei were counterstained with Hoechst stain.

Fig 2

Recognition of the E protein. (A) Western blots were prepared with gradient-purified DENV-2 particles, and blot strips were probed with hMAbs 4.8A, D11C, and 1.6D or anti-DENV capsid mMAb D2-C2 (41) under reducing and nonreducing conditions. Binding of hMAbs to DENV-2 proteins on the blot strips was detected at a PMT voltage of 400 V. Protein standards are indicated in kilodaltons. (B) Western blots were prepared with DENV-2 sE, and blot strips were probed with hMAbs 4.8A, D11C, 1.6D, and control mMAbs 4G2 and 3H5.1 under nonreducing conditions. Binding of hMAbs and mMAbs to DENV-2 sE on the blot strips was detected at a PMT voltage of 220 V.

Fig 3

Broad neutralizing activity. Focus-forming-unit reduction neutralization assays were performed by incubating DENV-1, -2, -3, and -4 with serial dilutions of sera from patients 8C and DA003 (A), hMAb 4.8A (B), hMAb D11C (C), and hMAb 1.6D (D) prior to infecting monolayers of LLC-MK₂ cells. IC₅₀s (in μg/ml) were determined graphically and were as follows: for hMAb 4.8A with DENV-1, 2.1 ± 1.1, DENV-2, >40, DENV-3, 2.4 ± 0.1, and DENV-4, >40; for hMAb D11C with DENV-1, 1.5 ± 0.1, DENV-2, 1.0 ± 0.4, DENV-3, 10.2 ± 0.8, and DENV-4, 1.6 ± 0.6; and for hMAb 1.6D with DENV-1, 1.5 ± 1.1, DENV-2, 0.2 ± 0.0, DENV-3, 0.5 ± 0.1, and DENV-4, 2.7 ± 0.8. The pooled data points show the means of at least two independent experiments with three replicates each. The error bars indicate standard deviations.

Fig 4

Antibody-dependent enhancement. Enhanced infection of Fc receptor-bearing K562 cells was measured by DENV-specific qRT-PCR following infection with DENV-1 (A), DENV-2 (B), DENV-3 (C), and DENV-4 (D) in the presence of hMAbs 4.8A, D11C, and 1.6D. Each data point is the mean of three replicates. The error bars indicate standard deviations.

Fig 5

Mechanism of neutralization. (A) Low-pH-activated virus-liposome fusion was measured using fluorescently labeled DENV-2 incubated with hMAbs 4.8A, D11C, and 1.6D. The fluorescence signal was normalized to the signal generated in the absence of hMAbs to calculate percent liposome fusion. EH21 is an irrelevant anti-HIV hMAb. (B) Intracellular fusion of DiD-labeled DENV-2 within endosomes leads to dequenching of DiD. Confluent monolayers of MA104 cells were infected with equivalent amounts of DENV-2 preincubated with or without 100 μg/ml hMAbs, as indicated. Intracellular structures at the site of fusion events fluoresce red. Cells were counterstained with DAPI to visualize nuclei. (C) Intracellular fusion levels were quantified after incubation of DENV-2 with different concentrations of hMAbs. Fluorescence levels were normalized to those of virus-only controls. (D) Total fluorescence of all bound DENV-2 was quantified by fully dequenching the cells. DENV-2 was incubated with 100 μg/ml of each hMAb. Fluorescence levels were normalized to those of virus-only controls. Heparan sulfate at 10 μg/ml, a known inhibitor of DENV binding, was used as a positive control for binding inhibition. For panels A, C, and D, each data point is the mean of three replicates. The error bars indicate standard deviations.

Fig 6

Coarse-level epitope mapping. (A) Western blots were prepared with DENV-2 sDI/II and sDIII, and blot strips were probed with 5 μg/ml of hMAbs 4.8A, D11C, 1.6D, and control mMAbs 4G2 and 3H5.1 under nonreducing conditions. Binding of antibodies to sDI/II on the blot strips was detected at a PMT voltage of 475 V or 562 V for hMAbs and mMAbs, respectively, whereas binding of both hMAbs and mMAbs to sDIII on blot strips was detected at a PMT voltage of 420 V. (B) A competition ELISA was used to determine whether hMAbs 4.8A, D11C, and 1.6D and mMAb 4G2 recognized overlapping epitopes on DENV-1 E protein. HMAb EH21 against HIV-1 ENV was used as a negative control. Unlabeled antibodies (shown on the x axis) were added to DENV-1 E protein-coated wells. Upon removal of unbound antibodies, the wells were probed with biotinylated antibodies as shown. (C) Antibody binding competition was measured using biolayer interferometry. Biosensor probes were coupled to hMAb 1.6D and subsequently incubated with either DENV-2 sE alone or sE complexed with hMAb 1.6D or control anti-HIV 1.7B or with mMAb 4G2 or 3H5.1.

Fig 7

Molecular-level epitope mapping. (A) Cells expressing DENV E mutants were fixed and immunostained with the indicated antibodies. Clones with reactivities of ≤25% relative to wild-type (WT) DENV-3 E were identified as critical for hMAb binding. The reactivities of mutant clones containing each critical residue with hMAbs 4.8A, D11C, and 1.6D and the control mMAb 1A1D-2 and human polyclonal serum (hPAb) are shown. The experiments were repeated three times, and standard deviations of quadruplicate wells are shown. (B) Critical residues for hMAbs 4.8A (W101, L107, and G109), D11C (W101 and G109), and 1.6D (W101 and G109) were visualized on a structure of DENV-3 E protein (Protein Data Bank accession code 1uzg [ 87]). DI, DII, and DIII are depicted in red, yellow, and blue, respectively, and the fusion loop (residues 98 to 109) is circled.