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      A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus

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          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 AC 50, 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 AC 50 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 AC 50 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.

          IMPORTANCE

          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.

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          Most cited references38

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          Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme.

          Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.
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            Antigen-specific memory B cell development.

            Helper T (Th) cell-regulated B cell immunity progresses in an ordered cascade of cellular development that culminates in the production of antigen-specific memory B cells. The recognition of peptide MHC class II complexes on activated antigen-presenting cells is critical for effective Th cell selection, clonal expansion, and effector Th cell function development (Phase I). Cognate effector Th cell-B cell interactions then promote the development of either short-lived plasma cells (PCs) or germinal centers (GCs) (Phase II). These GCs expand, diversify, and select high-affinity variants of antigen-specific B cells for entry into the long-lived memory B cell compartment (Phase III). Upon antigen rechallenge, memory B cells rapidly expand and differentiate into PCs under the cognate control of memory Th cells (Phase IV). We review the cellular and molecular regulators of this dynamic process with emphasis on the multiple memory B cell fates that develop in vivo.
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              Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages.

              An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles.
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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                4 August 2015
                Jul-Aug 2015
                : 6
                : 4
                : e01156-15
                Affiliations
                [ a ]Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
                [ b ]Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA
                [ c ]Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
                [ d ]Center of Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA
                [ e ]Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
                Author notes
                Address correspondence to Jonathan W. Yewdell, jyewdell@ 123456mail.nih.gov .

                G.M.F. and D.A. contributed equally to this work.

                Editor Gary J. Nabel, Sanofi

                This article is a direct contribution from a Fellow of the American Academy of Microbiology.

                Article
                mBio01156-15
                10.1128/mBio.01156-15
                4526714
                26242629
                039e8e2f-2151-4f59-a86b-a3ebd36a3ae7
                Copyright © 2015 Frank et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 10 July 2015
                : 14 July 2015
                Page count
                supplementary-material: 3, Figures: 6, Tables: 0, Equations: 0, References: 49, Pages: 11, Words: 9950
                Categories
                Research Article
                Custom metadata
                July/August 2015

                Life sciences
                Life sciences

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