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

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.