Assessing functional immunity to influenza infection by quantifying BCR binding avidities

PROJECT SUMMARY Activation, clonal expansion, and affinity maturation of antigen-specific B cells are considered the hallmarks of adaptive immunity; hence, the assessment of B cell responses is thought to provide correlates of immune protection. Antigen-specific B cell responses have traditionally been observed indirectly, by examining the serum antibody pool using ELISA, or in the case of influenza the hemagglutinin inhibition assay (HIA). Direct measurements of antibody-secreting cells have also been done using ELISPOT, or more recently by flow cytometry. However, there are currently no tools to assess the functionality of the non-antibody (Ab) secreting memory B cell (Bmem) pool. This is an important limitation as Bmem, more than the circulating Ab pool, contain cross-reactive and cross-protective cells that could respond to a challenge infection with a mutated pathogen variant, such as occurs during the seasonal yearly influenza surges. Knowing the extent to which Bmem cells exist and can bind to the original or emerging variants of a pathogen would greatly help, for example, with decisions about when to update vaccines to the circulating seasonal influenza or SARS-COV-2 strains. The primary objective of this proposal is to generate such a tool. Our recent work demonstrates the proof-of- concept that a simple microfluidic platform can capture the full avidity spectrum of antigen-specific B cells, and that the equilibrium binding avidity of the BCR correlates strongly with the binding affinity of the secreted antibody. We have dubbed this approach and technology the Shear-force Avidity-based Cell Selector (SACS). These findings provide the foundation of our central hypothesis: capture, separation, and quantification of peripheral B cells based on the force-dependent BCR binding avidity can be used as a measure of functional immune status. To test our hypothesis, and fully develop the SACS technology, the specific aims of this project are to: 1) enhance the design and optimize a microfluidic device with the capacity to capture and separate a complex pool of B cells based on BCR binding avidity to a defined antigen; and 2) characterize the relationship between binding avidity dynamics of influenza-specific peripheral B cells and immune status following influenza virus infection. Our platform will demonstrate the functionality of a rapid and easily adaptable system to evaluate the dynamics of the B cell response to influenza, but is completely adaptable to host of other pathogens such as SARS-Cov-2. Expected results would provide a new measurement platform (SACS) that would allow for the first time the rapid assessment of the range of functional BCR-antigen interaction strengths of individual B cells within the complex pools of peripheral B cells and B cell subsets to predict functional immunity.