title: Isolation of dengue virus-specific memory B cells with live virus antigen from human subjects following natural infection reveals the presence of diverse novel functional groups of antibody clones.
abstract: Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of <10 ng/ml. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein, including epitopes present only on the intact, fully assembled viral particle. We also isolated a number of serotype-cross-neutralizing MAbs, most of which recognized a region in E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.
  
  
  OBJECTIVE
  Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.
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: A Ruklanthi
authors.initials: AR
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 Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
authors.forename: Ramesh S
authors.initials: RS
authors.lastname: Jadi
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
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.
authors.forename: James E
authors.initials: JE
authors.lastname: Crowe
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D003429:Cross Reactions; D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000276:immunology*; D006801:Humans; D009500:Neutralization Tests; D014759:Viral Envelope Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D014759:Viral Envelope Proteins
references.citation: PLoS Negl Trop Dis. 2011 Jun;5(6):e1188
references.pmid: 21713020
references.citation: Sci Transl Med. 2012 Jun 20;4(139):139ra83
references.pmid: 22723463
references.citation: J Virol. 2008 Sep;82(17):8828-37
references.pmid: 18562544
references.citation: Nature. 2013 Apr 25;496(7446):504-7
references.pmid: 23563266
references.citation: Science. 2010 May 7;328(5979):745-8
references.pmid: 20448183
references.citation: Virology. 1994 Aug 1;202(2):885-90
references.pmid: 7518164
references.citation: Virology. 2009 Sep 15;392(1):103-13
references.pmid: 19631955
references.citation: Cell Host Microbe. 2010 Sep 16;8(3):271-83
references.pmid: 20833378
references.citation: J Virol. 2007 Dec;81(23):12816-26
references.pmid: 17881453
references.citation: Trends Microbiol. 2002 Feb;10(2):100-3
references.pmid: 11827812
references.citation: Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):1939-44
references.pmid: 24385585
references.citation: J Clin Microbiol. 2007 Nov;45(11):3777-80
references.pmid: 17804661
references.citation: Cell Host Microbe. 2010 Feb 18;7(2):128-39
references.pmid: 20153282
references.citation: EMBO Mol Med. 2014 Mar;6(3):358-71
references.pmid: 24421336
references.citation: PLoS One. 2009;4(4):e4991
references.pmid: 19337372
references.citation: Viruses. 2011 Dec;3(12):2374-95
references.pmid: 22355444
references.citation: J Virol. 2001 Aug;75(16):7769-73
references.pmid: 11462053
references.citation: J Virol. 2012 Mar;86(5):2665-75
references.pmid: 22171265
references.citation: J Infect Dis. 2013 Jun 15;207(12):1898-908
references.pmid: 23526830
references.citation: Nature. 1977 Feb 24;265(5596):739-41
references.pmid: 404559
references.citation: Proc Natl Acad Sci U S A. 2012 May 8;109(19):7439-44
references.pmid: 22499787
references.citation: J Clin Microbiol. 2005 Jul;43(7):3267-72
references.pmid: 16000446
references.citation: Virology. 2007 Sep 30;366(2):349-60
references.pmid: 17719070