The requirement of Toll-like receptor signaling in the induction of humoral and cell-mediated immune responses to the orally administered DukoralRTM vaccine.

摘要

Only a limited number of orally-administered vaccines have been licensed, such as the Dukoral® vaccine, comprised of killed whole-cell Vibrio cholerae and cholera toxin B subunit (CTB). Thus far, mechanistic details underlying the resulting protective immune generated by this vaccine, including the requirement of Toll-like receptor (TLR) signaling, remain largely unknown. Herein, I investigated the involvement of TLR signaling in the induction of humoral immune responses following oral and intramuscular immunization with Dukoral or its components by using TLR-2-, TLR-4-, MyD88- and Trif-deficient mice. I showed that wild type and all groups of TLR-deficient mice generated similar levels of V. cholerae- and CTB-specific IgG1 and IgG2c serum antibodies, and fecal IgA antibodies, following oral immunization with Dukoral, and with CTB alone. Additionally, the agglutinating activity of V. cholerae-specific antibodies was also found to occur independently of MyD88 signaling. However, intramuscular immunization with Dukoral, as well as with CTB alone, required MyD88 signaling for the induction of CTB-specific IgG1and IgG2c serum antibodies.;I also evaluated the involvement of TLR signaling in the induction of splenic cell-mediated immune responses following oral immunization with Dukoral. My results showed that CD4+ T-cell and CD19+ B cell proliferation occurred in a MyD88-dependent manner in response to stimulation by V. cholerae or CTB. In contrast, in response to CTB stimulation, Trif negatively regulated both CD4+ T-cell and CD19+ B-cell proliferation. Splenocytes from MyD88-/-, Trif-/-, TLR-2-/-, and TLR-4-/- mice were significantly inhibited in their ability to secrete IFN-γ in response to stimulation by V. cholerae. Furthermore, maturation of dendritic cells (DCs), as measured by increased cell-surface CD80, CD86, CD40, and MHCII expression and cytokine secretion was shown to occur in a MyD88-dependent manner in response to stimulation with Dukoral vaccine components. However, despite the impaired ability of MyD88-/- DCs to mature, MyD88-/- animals, and indeed all TLR-deficient animals tested, showed serum and fecal antibody responses comparable to those seen in wild-type animals. Taken together, my results suggest that humoral responses (antibody production and agglutinating ability), following oral immunization with Dukoral, were able to occur independently of TLR signaling and DC maturation. This TLR-independence in the generation of humoral responses was lost when the vaccine was administered parenterally. Cell-mediated immune responses (cell proliferation, DC maturation, and cytokine secretion) were found to be TLR-dependent.