title: Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay.
abstract: Dengue is a major public health problem globally. It is caused by four antigenically distinct serotypes of dengue virus (DENV1-4), and although serotype-specific and strongly neutralizing cross-reactive immune responses against the four DENV serotypes are thought to be protective, subneutralizing Abs can contribute to increased disease severity upon secondary infection with a different DENV serotype. Understanding the breadth of the immune response in natural DENV infections and in vaccinees is crucial for determining the correlates of protection or disease severity. Transformation of B cell populations to generate mAbs and ELISPOT assays have been used to determine B cell and Ab specificity to DENV; however, both methods have technical limitations. We therefore modified the conventional ELISPOT to develop a Quad-Color FluoroSpot to provide a means of examining B cell/Ab serotype specificity and cross-reactivity on a single-cell basis. Abs secreted by B cells are captured by an Fc-specific Ab on a filter plate. Subsequently, standardized concentrations of all four DENV serotypes are added to allow equal stoichiometry for Ag binding. After washing, the spots, representing individual B cells, are visualized using four fluorescently labeled DENV serotype-specific detection mAbs. This method can be used to better understand the breadth and magnitude of B cell responses following primary and secondary DENV infection or vaccination and their role as immune correlates of protection from subsequent DENV infections. Furthermore, the Quad-Color FluoroSpot assay can be applied to other diseases caused by multiple pathogen serotypes in which determining the serotype or subtype-specific B cell response is important.
authors: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720.
authors: Alexandros
authors: A
authors: Hadjilaou
authors: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720.
authors: Angela M
authors: AM
authors: Green
authors: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720.
authors: Josefina
authors: J
authors: Coloma
authors: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720 eharris@berkeley.edu.
authors: Eva
authors: E
authors: Harris
mesh_terms: D000330:Aedes; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology; D002478:Cells, Cultured; D003429:Cross Reactions / Q000276:immunology; D003715:Dengue / Q000276:immunology / Q000821:virology; D053059:Dengue Vaccines / Q000276:immunology; D003716:Dengue Virus / Q000145:classification* / Q000235:genetics / Q000276:immunology*; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D006801:Humans; D059010:Single-Cell Analysis; D014611:Vaccination; D014759:Viral Envelope Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D053059:Dengue Vaccines; D014759:Viral Envelope Proteins