| Local, protective immunity directed at pathogenic microorganisms is stimulated by primary infection or vaccination. Successful vaccination requires proper delivery of immunogens and effective immune stimulation. The described research evaluates the adjuvanticity of the Shigella invasin complex (Invaplex) in a mouse model. Intranasal immunization with Invaplex combined with either ovalbumin or Campylobacter FlaA enhanced antigen-specific antibody responses and cell-mediated immunity (CMI). The antigen-specific immune response was characterized by high levels of serum IgG1 and increased production of IL-4, IL-5, and IL-10, suggesting a Th2 response. Simultaneous delivery of Invaplex with ovalbumin resulted in the greatest enhancement of the ovalbumin-specific immune response; separation of the components yielded suboptimal immune responses. Delivery of Invaplex and antigen, separated by time, also led to enhanced immune responses, indicating an immunomodulatory activity of Invaplex not requiring physical association with the antigen.; The interaction of Invaplex and host cells in vitro indicated that Invaplex rapidly bound to eukaryotic plasma membranes and was internalized into the host cell cytoplasm by an actin-dependent endocytic process. MAbs directed at Shigella IpaB and IpaC proteins inhibited Invaplex internalization. After treatment with Invaplex, host cells did not leak cytoplasmic LDH or fail to exclude trypan blue. Internalized Invaplex trafficked through early endosomes, late endosomes, and the Golgi apparatus with eventual release into the cytosol. Invaplex also induced the uptake of heterologous molecules (proteins, plasmid DNA (pDNA)) indicating Invaplex possessed antigen-delivery capabilities. This suggested that Invaplex may serve as a mucosal adjuvant for DNA-based vaccines. Intranasal immunization with Invaplex and pDNA encoding the sta56 gene from Orienta tsutsugamushi induced Sta56-specific CMI. A DNA-prime-protein-boost immunization regimen enhanced both CMI and humoral Sta56-specific immune responses, demonstrating the capacity of Invaplex as a mucosal DNA vaccine adjuvant.; This dissertation research demonstrated that Invaplex enhanced humoral and cellular immunity to mucosally administered protein and DNA-encoded antigens. Invaplex functioned as an immunomodulatory adjuvant, able to enhance the immunogenicity of separately delivered antigens, and as an antigen-delivery system, capable of stimulating uptake of co-administered proteins and pDNA. The potent adjuvanticity and dual functionality of Invaplex warrant further development and clinical investigation for use with antigens from a wide spectrum of mucosal pathogens. |
