Functions of human C. difficile-specific memory B cell-derived monoclonal antibodies

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PROJECT SUMMARY / ABSTRACT (Project 2) Clostridioides difficile disease recurrence is a serious problem because severity increases with each round of infection and converts a regional enteric disease into a systemic fatal disease. We lack a good understanding of the immune response to infection, which arguably is lacking or mis-directed, as evidenced by recurrent infection. Serum IgG antibodies (Abs) that neutralize toxin B secreted by C. difficile (TcdB) is the best correlate of protection. Ideally, infection would stimulate primary toxin-neutralizing Ab responses as well as induce toxin- specific memory B cells (Bmem) that can respond rapidly to the pathogen and generate new Ab-secreting cells. However, in mouse models and in analysis of human Bmem, infection results in a response which is dominated by IgM+ cells, although some IgG+ and IgA+ cells are evident. Isolation and single cell barcoding of human TcdB- specific Bmem followed by sequencing and repertoire analysis revealed that IgG+ and IgA+ cells had undergone somatic hypermutation and had breadth of variable gene usage, representing several unique B cell clones. Production of monoclonal Abs (mAbs) from selected IgG1 gene sequences revealed moderate affinity for TcdB and poor TcdB neutralization in one in vitro assay. This work has guided us to a hypothesis that TcdB-specific IgG and IgA encoded by the C. difficile-induced human B cell memory compartment have variable capacity for toxin-neutralization. In Specific Aim 1 we will measure the impact of TcdB-specific Bmem-derived mAbs on the mechanisms of host cell intoxication. We will produce mAbs from gene sequences in our database and recruit new volunteers to expand the number of Bmem-repertoires. We will determine the impact of Bmem- derived mAbs on the mechanisms controlling host cell intoxication and provide a comprehensive view of the functions of human Bmem cell-encoded TcdB-specific Abs in individuals following C. difficile infection. In Specific Aim 2: We will test the ability of Bmem cell-derived mAbs to protect against a live pathogen challenge and determine mechanism of transport to the gut. We showed that the neonatal Fc receptor (FcRn) was required for delivery of immunization-induced circulating IgG to the gut and protection against C. difficile, whereas intraperitoneal delivery to recipient mice bypassed the FcRn requirement . We will deliver intraperitoneal mAb to B6 mice to determine if human Bmem-derived mAbs are protective in vivo. We will use FcRn-/- mice expressing the human FcRn and accessory molecule β2 microglobulin (hβ2M) transgenes (hFcRn:hβ2M ) to determine hFcRn dependence for protection by circulating mAb. Aim 2 will provide critical data on whether Bmem-encoded mAbs are protective in vivo and determine how they reach the gut. Project 2 has a high degree of relevance to public health. Our current understanding of the humoral immune response to C. difficile in human subjects lacks the necessary mechanistic insights to explain why patients are prone to recurrent infection. Our work will determine if the Bmem compartment insufficiently encodes protective Abs and also reveal the functional properties of protective versus non-protective Bmem cells and inform vaccination strategies.