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Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus

Nature Immunology volume 19, pages 1248–1256 (2018)Cite this article

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Abstract

Dengue virus is a major pathogen, and severe infections can lead to life-threatening dengue hemorrhagic fever. Dengue virus exists as four serotypes, and dengue hemorrhagic fever is often associated with secondary heterologous infections. Antibody-dependent enhancement (ADE) may drive higher viral loads in these secondary infections and is purported to result from antibodies that recognize dengue virus but fail to fully neutralize it. Here we characterize two antibodies, 2C8 and 3H5, that bind to the envelope protein. Antibody 3H5 is highly unusual as it not only is potently neutralizing but also promotes little if any ADE, whereas antibody 2C8 has strong capacity to promote ADE. We show that 3H5 shows resilient binding in endosomal pH conditions and neutralizes at low occupancy. Immunocomplexes of 3H5 and dengue virus do not efficiently interact with Fcγ receptors, which we propose is due to the binding mode of 3H5 and constitutes the primary mechanism of how ADE is avoided.

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Fig. 1: Neutralization and enhancement characteristics of the anti-DENV-2 antibodies 3H5 and 2C8.
Fig. 2: Binding of DENV immunocomplexes to FcγRs.
Fig. 3: Binding properties of 2C8 and 3H5.
Fig. 4: Crystal structures of 2C8–EDIII and 3H5–EDIII complexes.
Fig. 5: Epitope recognition by 3H5 and 2C8.
Fig. 6: Fab binding in the context of the mature virion.

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Data availability

The data supporting the findings of this study are available from the corresponding authors upon request. Structure factors and final refined coordinates of crystal structures have been deposited in the RCSB PDB (https://www.rcsb.org/pdb/) under the accession codes 6FLA (3H5 complex form 1), 6FLB (3H5 form2), and 6FLC (2C8).

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Acknowledgements

This work was supported by the Wellcome Trust, UK; the Newton-Medical Research Council, UK (MR/NO12658/2); the National Institute for Health Research Oxford Biomedical Research Centre funding scheme; the Thailand National Center for Genetic Engineering; and Biotechnology, the Academy of Finland; the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative; the Research Chair Grant from the National Science and Technology Development Agency (NSTDA), Thailand; and Faculty of Medicine Siriraj Hospital, Mahidol University, Grant Number (IO)R015936005. The OPIC electron microscopy facility was founded by a Wellcome Trust JIF award (060208/Z/00/Z) and is supported by a Wellcome Trust equipment grant (093305/Z/10/Z). The Wellcome Trust is also acknowledged for providing administrative support (Grant 075491/Z/04). G.R.S. is a Wellcome Trust senior investigator (095541/Z/11/Z and 203224/Z/16/Z). M.R. and A.F. were supported by Wellcome Trust fellowships (204703/Z/16/Z and 080721/Z/06/Z). We thank A. Siebert for support with electron microscopy; T. Prommool and C. Tawilert for scale-up and purification of monoclonal antibodies; P. Keelapang for good advice on NT and ADE assays; and D. Bitto for assistance with cryo-EM sample preparation. We also thank the staff of the Diamond Light Source (proposal MX8423), and ESRF, K. Harlos, and T. Walter, for assistance with crystallization; and D. Stuart for scientific discussion.

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  1. These authors contributed equally: Max Renner, Aleksandra Flanagan, Wanwisa Dejnirattisai.

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Max Renner, Aleksandra Flanagan, Juha T. Huiskonen & Jonathan M. Grimes

  2. Nuffield Department of Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Wanwisa Dejnirattisai, Piyada Supasa, Wiyada Wongwiwat, Alison Cowper, Claire M. Midgley, Juthathip Mongkolsapaya & Gavin R. Screaton

  3. Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Chunya Puttikhunt, Kriangkrai Chawansuntati, Thaneeya Duangchinda & Prida Malasit

  4. Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

    Chunya Puttikhunt, Kriangkrai Chawansuntati, Prida Malasit & Juthathip Mongkolsapaya

  5. Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, Chiang Mai University, Chiang Mai, Thailand

    Watchara Kasinrerk

  6. Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

    Watchara Kasinrerk

  7. Science Division, Diamond Light Source Ltd, Didcot, UK

    Jonathan M. Grimes

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Contributions

Experiments were conceived and designed by A.F., M.R., W.D., J.M., J.T.H., J.M.G., and G.R.S. Experiments were performed by A.F., M.R., W.D., P.S., W.W., K.C., T.D., A.C., and C.M.M. The data were analyzed by A.F., M.R., W.D., C.M.M., J.M., J.T.H., J.M.G., and G.R.S.. The paper was written by A.F., M.R., J.M., J.T.H., J.M.G. and G.R.S. C.P., W.K. and P.M. provided antibodies.

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Correspondence to Juthathip Mongkolsapaya, Gavin R. Screaton or Jonathan M. Grimes.

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Integrated supplementary information

Supplementary Figure 1 ADE in K562 cells and Fcγ receptor subtype dependence.

a,b, Enhancement properties of 3H5 (orange) and 2C8 (blue) in K562 cells. DENV2 strains NGC and 16681 (as indicated) were incubated with antibody before infecting K562 cells for enhancement assays. Supernatants of K562 cultures were harvested after 4 d and titrated onto Vero cells for a focus-forming assay. c,d, Effect of Fcγ receptor blocking on enhancement. Anti-FcγR antibodies (as indicated) were used to block Fc receptors during incubation of antibody–virus immunecomplexes with U937 cells or K562 cells to assess routes of enhancement. Supernatants of the cultures were harvested after 4 d and titrated onto Vero cells for a focus-forming assay.

Supplementary Figure 2 Comparison of the enhancement and neutralization properties of humanized 2C8-LALA.

a–d, LALA mutations were introduced into humanized 2C8 (Hu2C8) and enhancement (a,b) and neutralization (c,d) characteristics were assessed for DENV2/NGC and DENV2/16681 as indicated. In all experiments, DENV2 strains NGC and 16681 were incubated with antibody before infecting Vero or U937 cells for neutralization or enhancement assays, respectively. Supernatants of U937 cultures were harvested after 4 d and titrated onto Vero cells for a focus-forming assay. The data are shown as means ± s.e.m. from three independent experiments.

Supplementary Figure 3 Representative electron density.

a, Stereo diagram of the interaction between the EDIII FG loop (blue) and the 3H5 heavy chain (gray) and light chain (green). The interfacial waters involved in water-mediated hydrogen bonds are shown in red. The hydrogen bonds and salt bridges are marked with black dashed lines. b, Stereo diagram of the interaction between the N-terminal region of EDIII (blue) and the 2C8 Fab heavy chain (gray) and light chain (teal).

Supplementary Figure 4 CDRs of 2C8 and 3H5.

a, 2C8 variable domains (VL in light green and VH in dark green) bound to EDIII (blue) in cartoon representation. The complementarity-determining regions (CDRs) for the light chain (L1–L3) and heavy chain (H1–H3) are indicated. b, 3H5 variable domains (VL in light orange and VH in orange) bound to EDIII (blue). The CDRs are indicated. c, CDR-L1 of 3H5 intimately interacts with the surface of EDIII (blue) with the key residue F32 (shown as sticks) located at a central position.

Supplementary Figure 5 Comparison of EDIII residues targeted by 3H5 and quaternary epitope antibodies 2D22 and C8-EDE.

The general architecture of EDIII is shown on the left and important regions are labeled. Residues engaged by the lateral ridge antibody 3H5 are displayed as orange sticks. Residues on EDIII that are included in the epitope of quaternary antibodies 2D22 and C8 EDE are shown as green and yellow sticks, respectively.

Supplementary Figure 6 Cryo-electron microscopy analysis of DENV-2 incubated with 2C8 Fabs at room temperature.

a, Representative cryo-EM micrograph of DENV-2 particles incubated with 2C8 Fabs at room temperature showing virions that are partially occupied with Fabs. b, Comparison of the radial density profiles of particles incubated with 2C8 at room temperature (black curve) and at 37 °C (red curve) showing a distinct increase in Fab density (indicated by the white arrow). Radial density profiles were calculated from 2D averages of centered particles using IMAGIC.

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Renner, M., Flanagan, A., Dejnirattisai, W. et al. Characterization of a potent and highly unusual minimally enhancing antibody directed against dengue virus. Nat Immunol 19, 1248–1256 (2018). https://doi.org/10.1038/s41590-018-0227-7

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