Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement Melissa A. Edeling 1 , S. Kyle Austin 1 , Bimmi Shrestha 1 , Kimberly A. Dowd 2 , Swati Mukherjee 2 , Christopher A. Nelson 1 , Syd Johnson 3 , Manu N. Mabila 4 , Elizabeth A. Christian 4 , Joseph Rucker 4 , Theodore C. Pierson 2 , Michael S. Diamond 1,5,6 , Daved H. Fremont 1,6,7 * 1 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 Viral Pathogenesis Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 3 MacroGenics, Rockville, Maryland, United States of America, 4 Integral Molecular, Philadelphia, Pennsylvania, United States of America, 5 Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America, 6 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 7 Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, United States of America Abstract We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 A ˚ resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ,20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface. Citation: Edeling MA, Austin SK, Shrestha B, Dowd KA, Mukherjee S, et al. (2014) Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement. PLoS Pathog 10(4): e1004072. doi:10.1371/journal.ppat.1004072 Editor: Fe ´lix A. Rey, Institut Pasteur, France Received August 27, 2013; Accepted March 3, 2014; Published April 17, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Pediatric Dengue Vaccine Initiative, the intramural program of NIAID, and NIH grants R01-AI077955, U01-AI061373, U54 AI057160 (Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research), and NIAID contracts HHSN272200700058C and HHSN272201200026C (Center for Structural Genomics of Infectious Disease) and HHSN272200900055C (B Cell Epitope Discovery and Mechanisms of Antibody Protection). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: SJ is an employee and MSD is a paid consultant for MacroGenics. MNM, EAC and JR are employees of Integral Molecular. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. * E-mail: fremont@wustl.edu Introduction Dengue virus (DENV) infection in humans causes symptoms ranging from a mild febrile illness to a severe and sometimes fatal disease. Over 3.6 billion people globally are at risk for DENV infection, with an estimated 390 million infections annually and no currently approved vaccine or antiviral therapy [1]. DENV belongs to the Flaviviridae family of medically important positive- stranded RNA viruses. Within the DENV serocomplex, there is significant diversity, including four serotypes (DENV-1, -2, -3, and 4) that differ at the amino acid level of the envelope (E) protein by ,25 to 40 percent and multiple genotypes within a serotype that vary by up to ,3 percent [2,3]. A humoral response against DENV infection is believed to contribute to lifelong immunity against challenge by the homol- ogous serotype. In comparison, protection against a heterologous DENV serotype infection is more transient (,6 months to two years) [4,5], allowing re-infection and disease to occur with a heterologous serotype in hyper-endemic areas of the world. Estimates suggest that greater than 90% of severe cases occur during secondary infection with a heterologous DENV serotype, possibly because sub-neutralizing amounts of cross-reactive antibody facilitate viral entry into myeloid cells expressing Fc-c receptors, a phenomenon termed antibody-dependent enhance- ment of infection (ADE) [6]. Antibody-mediated protection against homologous DENV infection correlates with a neutralizing antibody response directed predominantly against the viral E protein [7]. The ectodomain of E is comprised of three domains: domain I (DI), a central nine-stranded b-barrel that connects domain II (DII), which contains the fusion peptide at its distal end, and an immunoglobulin-fold like domain III (DIII) [6–9]. Although neutralizing antibodies have been mapped to all three domains of the E protein, many potently inhibitory anti-DENV mouse MAbs map to DIII [10–17], specifically to the lateral ridge PLOS Pathogens | www.plospathogens.org 1 April 2014 | Volume 10 | Issue 4 | e1004072