Structural basis of Chikungunya virus inhibition by monoclonal antibodies

Abstract

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Keywords: antibody; chikungunya virus; cryo-EM; epitope.

Fig. 1.

CHK-124 and CHK-263 protect mice against CHIKV diseases and dissemination. (A–J) CHK-124 and CHK-263 mAbs reduced foot swelling and the viral RNA levels in ankles, muscles, blood, and spleen of CHIKV-infected wild-type (A–E) or congenic FcRγ^−/− (F–J) mice. Four-week-old wild-type or FcRγ^−/− C57BL/6 mice were treated with 100 µg of CHK-124, CHK-263, or an isotype control (WNV E60) 1 d before infection with 10³ FFU of CHIKV. (A and F) Foot swelling was measured at 3 dpi. Horizontal line indicates mean values (n = 5 to 10/group; two experiments; one-way ANOVA with a Tukey’s posttest; ****P < 0.0001). (B and G) Ipsilateral ankle, (C and H) serum, (D and I) spleen, and (E and J) contralateral ankle and gastrocnemius (calf) muscle were harvested 3 dpi, and viral RNA levels were determined by qRT-PCR. Bars indicate median values (n = 5 to 10/group); two experiments; Kruskal–Wallis with a Dunn’s posttest; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Each symbol in this figure represents data from an individual mouse.

Fig. 2.

CHK-124 and CHK-263 target multiple pathways in the CHIKV infection cycle. In general, CHK-124 inhibitory activities were more dramatically reduced when bivalency of IgG was abolished by the use of Fab fragments compared to CHK-263. (A) CHK-124 and CHK-263 IgGs are highly neutralizing in both pre- and postattachment neutralization assays, whereas their Fabs have weaker activities. PRNT₅₀ values indicating the antibody concentration that neutralized 50% of the plaque-forming units was determined by curve fitting using nonlinear regression in GraphPad Prism v8.0. (B) CHK-124 IgG induced virus aggregation, which correlates with its neutralization profile, whereas CHK-263 does not cause virus aggregation. The hydrodynamic size of CHIKV:CHK-124 IgG and CHIKV:CHK-263 IgG complexes in a different IgG:virion molar ratio was measured by dynamic light scattering (black curve). The neutralization profile of these CHIKV:IgG complexes (red curve) is shown as a neutralization index (right y axis) calculated as the log₁₀ fold reduction of the virus titer compared to the virus-only control. (C) Both CHK-124 and CHK-263 IgG and Fab prevent virus from attaching to cells if antibody:CHIKV complex is formed before addition to cells. Isotype IgG/Fab and no antibody controls were included. (D) Virus:liposomal membrane fusion assays showed that both IgGs of CHK-124 and CHK-263 inhibit virus:liposomal membrane fusion at pH 5.5, similar to the positive control (dethylpyrocarbonate [DEPC]). When Fab fragments were used, the inhibitory effect of CHK-124 was abolished whereas that of CHK-263 Fab was maintained. The extent of fusion was calculated as the percentage of the fluorescence emission before adding Triton X-100 to the full fluorescence emission after adding Triton X-100. (E) CHK-124 has stronger inhibitory activity of viral egress than CHK-263. (A–E) Data are the mean ± SEM from at least three independent experiments. Significance was determined by one-way ANOVA with Dunnett’s posttest compared to isotype control. (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3.

The cryo-EM maps of CHIKV complexed with Fab fragment of CHK-124 or CHK-263. (A and B) The cryo-EM density maps of CHIKV:CHK-124 Fab (A) and CHIKV:CHK-263 Fab (B) were determined to 5.2- and 4.7-Å resolutions, respectively. Their surface and a cross-section of a quarter of cryo-EM density maps are shown in the Left and Right panels, respectively. The black triangles represent an icosahedral asymmetric unit, and their five-, three-, and two-fold vertices are indicated. The E1-E2 heterodimers in one icosahedral asymmetric unit are colored as blue. (C and D) Top (Left) and side (Right) view of the binding of CHK-124 Fab (C) and CHK-263 Fab (D) to an asymmetric unit of CHIKV surface E1-E2 proteins. CHK-124 Fabs bind to all individual E1-E2 heterodimers within an asymmetric unit in an orientation perpendicular to the virus lipid envelope surface. CHK-263 Fabs lie laterally to the virus surface binding to three of the four E1-E2 (molecules A–C but not D). The Fab A (dashed circle) density is poor, suggesting partial occupancy around the five-fold vertex. E1:E2 heterodimers are shown as ribbons and the Fab molecules as surface representations. CHK-124 and CHK-263 Fab molecules are colored in gray and pink, respectively. The CHIKV E1, E2, and capsid proteins are colored in yellow, green and red, respectively. Vertices are indicated. (E) Localized reconstruction of subregions around the five-fold vertices in the CHIKV:CHK-263 Fab-complexed structure (cyan circle in B, Left). Results showed five different classes of the subparticles. All indicate that at most only two Fabs can bind around the five-fold vertex and are unable to bind to two E1-E2 heterodimers located right next to each other. The Fab densities are colored as hot pink, and the E1-E2 heterodimers bound by these Fabs are colored in dark green. (Right) The averaged map of these classes after they have been rotationally aligned to each other at 4.5-Å resolution.

Fig. 4.

Epitopes bound by CHK-124 Fab and CHK-263 Fab. (A) Binding mode of CHK-124 Fab (Top) and CHK-263 Fab (Bottom) to the ectodomains of an E1-E2 heterodimer. Epitopes of CHK-124 and CHK-263 are shown as blue and pink spheres, respectively, on an E1-E2 heterodimer (Middle and Right). (Right) Zoom-in view of the epitope with strands labeled. (B) Open book representation showing the electrostatic potential of the interacting interface between the epitope and paratope of CHIKV:CHK-124 (Top) and CHIKV:CHK-263 Fab (Bottom) structures. Positive, negative, and neutral charged residues are colored in blue, red, and white, respectively. Cyan dashed lines indicate the boundaries of the antibody heavy and light chains in the paratope and also its corresponding binding epitope. Residues at the interacting interface are indicated.

Fig. 5.

The cryo-EM structure of CHIKV complexed with CHK-263 IgG. (A) Surface of the cryo-EM structure of CHIKV complexed with CHK-263 IgG. E1, E2, and IgG are colored in green, yellow and magenta, respectively. (B) The cryo-EM map of the localized reconstruction of subregions around the two-fold (quasi-six-fold) vertex of CHIKV:CHK-263 IgG complex, displayed at different contour levels. The densities corresponding to the Fabs of the IgG are colored in magenta, while their bound E1-E2 heterodimers are colored in dark green. Blue densities indicate two hinge densities each from an IgG. We therefore detected two IgGs bound around the two-fold vertex. (C) Zoom in side view of the bound IgGs. Black arrows pointing to the two hinges (in blue) each from an IgG. Weak densities corresponding to the Fc regions are colored in gray. (D) Two possible arrangements of the two IgGs around the two-fold vertex. The densities of two Fabs possibly from an IgG are in the same color. Distance between the adjacent Fab molecules of CHK-263 IgG is indicated in red fonts above the red lines. Measurement of an IgG crystal structure (PDB ID: 1IGT) shows that the distance between two Fabs in an IgG is within 87 Å.

Fig. 6.

Superposition of the CHIKV receptor Mxra8 onto the cryo-EM structures of the CHIKV:Fab complexes. (A and B) Superposition of the CHIKV receptor Mxra8 to the CHK-124 Fab (A) and CHK-263 Fab (B) structures within an asymmetric unit (Left). Zoom-in side views of E1-E2 heterodimer (Right) show that the binding of CHK-124 may clash with Mxra8, whereas CHK-263 Fab and Mxra8 can bind simultaneously to the E1-E2 heterodimer. E1: yellow ribbons; E2: green ribbons; Mxra8: blue surfaces; CHK-124: gray surfaces; CHK-263: pink surfaces. (C) CHK-124 IgG can strongly inhibit virus from binding to Mxra8 whereas CHK-263 IgG has only moderate activity. Blocking of Mxra8-Fc binding to CHK-124 or CHK-263 complexed CHIKV was determined by competition ELISA. CHIKV VLPs were captured with CHK-152 and CHK-166 before addition of the CHIKV mAbs or Mxra8-mouse-Fc followed by Mxra8-hu-Fc (human Fc). Black circles indicate Mxra8-hu-Fc binding to CHIKV VLP in the absence of anti-CHIKV mAbs. A rightward shift of the curve indicates competition of CHIKV mAbs with Mxra8-mFc for binding to CHIKV. Data are the mean ± SEM from two independent experiments performed in duplicate.