Student Theses and Dissertations

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Victora Laboratory

Abstract

Repeated exposure to viruses or their antigens elicits anamnestic antibody responses that produce antibodies that are of greater magnitude and affinity compared to those induced after primary exposure to antigen.The anamnestic nature of the response is a result of the recall of memory B cells (MBCs) that have undergone clonal expansion and affinity maturation in germinal centers(GCs) during the primary response. Upon antigen re-encounter (“boosting”), MBCs, aided by enhanced help from memory T cells, efficiently differentiate into antibody-secreting plasma cells. At the same time, boosting induces recall GCs that could in principle either further affinity mature primary-derived MBCs or engage naïve B cells with potentially new epitope-specificities. The balance between these two possibilities is important for the rational design of vaccine strategies that can induce broadly neutralizing antibodies against mutating viruses like HIV-1, influenza A virus and SARS-CoV-2. Moreover, it is important in the context of a serum phenomenon termed antigenic imprinting or “original antigenic sin” (OAS), in which the antibody response is thought to repeatedly derive from the first cohort of B cells engaged by primary antigenic exposure, at the expense of inducing de novo antibodies against related antigens. Recall antibody responses therefore emerge from a complex interplay of B cell clones with varying degrees of specificity, affinity maturation and via intercalating MBC stages. The response is further affected by T cell help and potentially shaped by the competition of B cells with pre-existing antibodies for access to antigen (antibody-mediated feedback). Our studies, employing various prime-boost models in genetic fate-mapping mice, address multiple aspects of the recall B cell and antibody response. First, using cellular fate-mapping in which we mark the primary-cohort B cells and their MBC progeny with fluorescent proteins, we addressed the contribution of MBC clones to the recall response. We found that recall responses are characterized by a clonality bottleneck that restricts the responding clones to few dominant ones out of the vast number of MBC clones generated throughout the primary response. These selected “winner clones” were generally derived from relatively high-affinity germline precursors compared to the large diversity of MBC clones that was not reengaged detectably by boosting. We also found that further diversification of MBCs in recall GCs does occur, but at very low frequency. Instead, recall GCs are composed predominantly of a clonally diverse repertoire of naïve-derived B cells that did not undergo prior affinity maturation. Despite this potential of recall GCs to induce de novo antibody responses, we found that these antibodies were suppressed in the serum of newly-generated “molecular fate-mapping” mice repeatedly boosted with the same antigen. Instead, we found that recall antibodies were almost exclusively derived from the primary cohort of B cells, even after three or four antigen exposures. However, this “primary addiction”, with OAS-type suppression of de novo responses, decreased upon boosting with variant antigens as a function of antigenic distance. These de novo antibodies targeted variant-specific epitopes not covered by primary-derived antibodies, consistent with antibody-mediated feedback. Thus, whereas recall GCs contain mostly naïve-derived B cells, these only contribute to serum antibody when the antigenic distance is sufficient, and instead recall antibodies tend to result from primary addiction. Finally, we set out to elucidate the potential effects of antibody-mediated feedback and memory T cell help on the differential reliance that recall GC and antibody responses have on MBCs. We show that this schism is at least partly explained by a marked decrease in the ability of recall GC B cells to detectably bind antigen. Variant priming and plasma cell ablation experiments show that this decrease is largely due to suppression by pre-existing antibody, whereas hapten-carrier experiments reveal that increased memory T cell help allows B cells with undetectable antigen binding to access GCs. We propose a model in which antibody-mediated feedback steers recall GC B cells away from previously targeted epitopes, thus enabling tailored targeting of viral escape epitopes. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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