DENGUE VIRUS. Cryo-EM structure of an antibody that neutralizes dengue virus type 2 by locking E protein dimers

Abstract

There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)-specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo-electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 "locks" two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

Fig. 1. Prophylactic and therapeutic efficacy of HMAb 2D22 and 2D22-LALA in DENV2-inoculated mice

(A) Prophylactic studies. AG129 mice injected with HMAb 2D22 24 hours before sublethal challenge with DENV2 strain D2S10 showed significant reduction in serum viremia and bone marrow viral load, compared to the control mice receiving IgG1 isotype control. The DENV2 limit of detection (LOD) is indicated by the dashed line. Significance was determined using a two-tailed Wilcoxon rank-sum test (*P < 0.05). Shown is one of two representative experiments, with n = 4 mice per group. (B) Therapeutic efficacy after high-dose-DENV2-lethal infection. AG129 mice were first inoculated with DENV2 D2S10, and then 20 µg of HMAb 2D22-LALA was administered 24 hours later. Chimeric human-mouse MAb E60-N297Q or IgG1 isotype controls were used to treat control animals. Shown are the combined results from two independent experiments, with n = 9, 8, or 4 mice for the IgG1, 2D22-LALA, or E60-N297Q groups, respectively. Significance was determined using the Mantel-Cox log-rank test (P < 0.001, 2D22-LALA versus IgG1). (C) Therapeutic efficacy after ADE-DENV2-lethal infection. AG129 mice were administered a lethal enhancing dose of anti-DENV1 serum 24 hours before inoculation with DENV2 D2S10. The indicated MAb was delivered intraperitoneally 24 hours after inoculation. The results from two independent experiments are shown, with n = 8 mice for the IgG1, 2D22-LALA (20 or 50 µg) and E60-N297Q (20 µg) groups, n = 4 for the group receiving 50 µg of E60-N297Q, and n = 3 for E87-N297Q. Mice receiving 20 or 50 µg of 2D22-LALA displayed a significant level of protection compared to IgG1 control mice (P < 0.001, Mantel-Cox log-rank test).

Fig. 2. The 6.5 Å resolution cryo-EM structure of 4° C-2D22-PVP94/07

(A) The surface (left) and cross section (right) of the cryo-EM map. One icosahedral asymmetric unit is indicated by a white triangle. (B) Three Fab 2D22 molecules bind per asymmetric unit. The three individual E proteins in an asymmetric unit are labeled as molecules A, B, and C. The same molecules from an adjacent asymmetric unit in a raft structure are labeled as A′, B′, and C′. DI, DII, and DIII of E protein are colored in red, yellow, and blue, respectively. (C) The 2D22 epitopes on an E protein raft. The epitopes on molecules A, B, and C are largely similar; however, that on molecule A also has some interactions with adjacent E proteins. The DI, DII, and DIII of the surrounding E proteins are colored in light gray, gray, and black, respectively. The residues that interact with heavy and light chains of the Fab are shown as violet and cyan spheres, respectively. Additional residues from adjacent E protein dimers that formed part of the epitope are shown as green spheres. The boundary of each epitope is indicated with a light blue dotted circle. (D) The glycan loop (residues 144 to 157) on one E protein moves away from the fusion loop of the opposite E protein in the dimer when Fab is bound. The E protein of the uncomplexed DENV2 (gray) is superimposed onto the E protein of the complexed structure.

Fig. 3. Cryo-EM structure of BF-37°C-2D22-NGC (Class II)

(A) Cryo-EM map of BF-37°C-2D22-NGC (Class II) (left) and the fitted structure of the variable region of the Fab complexed with E dimers on a raft (right). The Fab 2D22 heavy and light chains are colored in violet and cyan, respectively. (B) (i) The E proteins of BF-37°C-2D22-NGC are on a higher radius than those of 4°C-2D22-NGC or DENV2(NGC) stage 1. (ii) The E protein arrangement of BF-37°C-2D22-NGC is more similar to that of the 37°CDENV2(NGC) (stage 3) structure (top). The A-C′ dimer of BF-37°C-2D22-NGC is on a slightly higher radius, whereas the B-B′ are located lower when compared to the stage 3 NGC structure. (C) Postulated movement of the Fab-E protein complex from the 4°C-2D22-NGC to the BF-37°C-2D22-NGC structures. The partial lifting and rotation of the A-C dimers would knock off the bound Fab on the B-B′ dimer (zoomed-in panel). This may then be followed by the rotation of the B-B′ dimer.

Fig. 4. The cryo-EMmap of AF-37°C-2D22-NGC

(A) Surface of the cryo-EM map and (B) its central cross section. Fabs bound near the five- and threefold vertices are indicated by black and white arrows, respectively.