title: Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site.
abstract: The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells.
  
  
  OBJECTIVE
  Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells.
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Smith
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
authors.forename: Usha K
authors.initials: UK
authors.lastname: Nivarthi
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
authors.forename: Ruklanthi
authors.initials: R
authors.lastname: de Alwis
authors.affiliation: The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Nurgun
authors.initials: N
authors.lastname: Kose
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Gopal
authors.initials: G
authors.lastname: Sapparapu
authors.affiliation: The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Bombardi
authors.affiliation: Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Kahle
authors.affiliation: Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Pfaff
authors.affiliation: Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Sherri
authors.initials: S
authors.lastname: Lieberman
authors.affiliation: Integral Molecular, Inc., Philadelphia, Pennsylvania, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Doranz
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA james.crowe@vanderbilt.edu aravinda_desilva@med.unc.edu.
authors.forename: Aravinda M
authors.initials: AM
authors.lastname: de Silva
authors.affiliation: Department of Pediatrics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA james.crowe@vanderbilt.edu aravinda_desilva@med.unc.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Crowe
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000914:Antibodies, Viral / Q000378:metabolism*; D019067:Antibody-Dependent Enhancement; D003716:Dengue Virus / Q000187:drug effects* / Q000502:physiology; D018604:Epitope Mapping; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus