title: A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus.
abstract: Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design.
  
  
  OBJECTIVE
  Though it was first described 50 years ago, little is known about how antibody affinity maturation contributes to immunity. This question is particularly relevant to developing more effective vaccines for influenza A virus (IAV) and other viruses that are difficult vaccine targets. Limitations in methods for characterizing antigen-specific B cells have impeded progress in characterizing the quality of immune responses to vaccine and natural immunogens. In this work, we describe a simple flow cytometry-based approach that measures both the number and affinity of IAV-binding germinal center B cells specific for the IAV HA, the major target of IAV-neutralizing antibodies. Using this method, we showed that the route and form of immunization significantly impacts the quality and quantity of B cell antibody responses. This method provides a relatively simple yet powerful tool for better understanding the contribution of affinity maturation to viral immunity.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Frank
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Davide
authors.identifier: http://orcid.org/0000-0002-5256-1972
authors.initials: D
authors.lastname: Angeletti
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: William L
authors.initials: WL
authors.lastname: Ince
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Gibbs
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Surender
authors.initials: S
authors.lastname: Khurana
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Adam K
authors.initials: AK
authors.lastname: Wheatley
authors.affiliation: Center of Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Edward E
authors.initials: EE
authors.lastname: Max
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Adrian B
authors.initials: AB
authors.lastname: McDermott
authors.affiliation: Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Hana
authors.initials: H
authors.lastname: Golding
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
authors.forename: James
authors.initials: J
authors.lastname: Stevens
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA jyewdell@mail.nih.gov.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D001402:B-Lymphocytes / Q000737:chemistry* / Q000276:immunology* / Q000502:physiology; D002454:Cell Differentiation; D005434:Flow Cytometry / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D009980:Influenza A virus / Q000276:immunology*; D007273:Injections, Intramuscular; D051379:Mice; D011947:Receptors, Antigen, B-Cell / Q000032:analysis*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011947:Receptors, Antigen, B-Cell