Innate Immune Recognition of Conserved Structural Features of Bacterial Flagellin Shapes Host Antibody Responses

Thesis (Ph.D.)--University of Washington, 2014Within this dissertation, we have determined how components of the host's innate immune system and structural features of flagellin, a pathogen associated molecular pattern (PAMP) and the major structural component of bacterial flagella, determine isotype specific antibody responses. Bacteria use flagella for chemotaxis toward energy and nutrient resources. For Salmonella enterica serovar Typhimurium (S. Typhimurium) the primary structural protein comprising the flagella is flagellin, which is encoded by the genes, fljB and fliC. The primary flagellin molecule, FliC, has been crystallized revealing that its structure consists of four domains: D0, D1, D2 and D3. D0 is recognized by Naip5 and Naip6 (Naip5/6), which initiates the assembly of the inflammasome and caspase-1 activation. D1 is recognized by Toll-like receptor 5 (TLR5), which signals through adaptor protein MyD88 to initiate MAPK signaling and NF-κB activation. Using purified flagellin from S. Typhimurium, we dissected the contribution of innate flagellin recognition pathways to promote antibody responses towards flagellin and co-administered ovalbumin in mice. We demonstrate IgG2c responses towards flagellin were TLR5- and inflammasome-dependent; IgG1 was the dominant isotype and partially TLR5- and inflammasome-dependent. In addition, a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. To address how flagellin triggers the TLR5-, inflammasome-, and MyD88-independent pathway, we turned our attention to the flagellin molecule's highly conserved recognition sites located on domains D0 and D1, and its hypervariable D2 and D3 (D2/D3) domains. To determine how FliC's structural features affect the TLR5-, inflammasome-, and MyD88-independent pathway, we destroyed the D0 (FliC-C) or the D1 (FliCTLR5) recognitions sites, or deleted the D2/D3 domain (FliCD0/D1), and performed primary and secondary immunizations with C57BL/6 mice. Results from FliC-C, FliCTLR5, FliCD0/D1 immunized mice indicate that primary antibody responses towards flagellin are strictly dependent on FliC's D2/D3 domain. Robust secondary antibody responses in mice require TLR5 and caspase-1, or MyD88, and FliC's D2/D3 domain. This is the first formal demonstration that flagellin's D2/D3 domain is required for induction of primary antibody responses and robust secondary antibody responses. The compilation of our results identifies a novel recognition pathway for bacterial flagellin that plays a significant role in flagellin's immunogenicity. Flagellin also works as an adjuvant when co-administered with ovalbumin, but only promotes moderate IgG1 anti-OVA responses following secondary immunizations. In contrast to flagellin's modest adjuvancy towards co-administered antigens, we demonstrate that covalent linkage of antigens to flagellin significantly enhances the antigen's immunogenicity. By coupling antigens to flagellin, we tested several flagellin-HIV fusions and identified one construct that was capable of inducing antibodies specific for the 4E10 epitope of HIV's gp41 membrane proximal external region (MPER). These results establish a flagellin vaccine platform that may be exploited to enhance the immunogenicity of poorly immunogenic epitopes to combat debilitating diseases such as influenza, tuberculosis, malaria, and HIV