Production and Characterization of Enterobactin-specific Antibodies Induced by Novel Enterobactin Conjugate Vaccines
Date of Award
Master of Science
Jeremiah Johnson, Oudessa Kerro Dego
Enterobactin (Ent)-mediated high affinity iron acquisition is critical for Gram-negative bacteria to survive in the host. Given the bacteriostatic effect of lipocalin resulting from its potent Ent-binding ability, immune intervention directly targeting Ent is promising for iron-dependent pathogen control. Moreover, passive immunization with Ent-specific egg yolk antibody (IgY) is a promising alternative to antibiotics to prevent and treat bacterial infections. In the first study, using a simple method, we successfully conjugated purified Ent to different carriers including keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and CmeC, a vaccine candidate for Campylobacter control. Subcutaneous immunization of rabbits with the KLH-Ent conjugate triggered strong systemic IgG immune response with up to 16,384 fold increase in IgG titer directed against whole conjugate and up to 4,096 fold increase in the level of specific anti-Ent IgG. The Ent-specific IgG also displayed exceptional reactivity to ferric Ent, linear trimer of Ent, and different salmochelins. Growth assays further demonstrated the Ent-specific antibodies significantly inhibited Ent-dependent growth of Campylobacter spp. and E. coli. In the second study, using different vaccination regimens, we immunized laying hens with different Ent conjugate vaccines to produce Ent-specific egg yolk IgY. In the first trial, Barred Rock layers (n=3) were intramuscularly immunized with the KLH-Ent conjugate vaccine, which triggered significant immune response in layers, leading to moderately increased titers of Ent-specific IgY in serum (up to 8 folds) and yolk (up to 2 folds). In the second trial, Rhode Island Red layers were subcutaneously immunized with specific Ent conjugates (KLH-Ent, BSA-Ent, or CmeC-Ent; n=2 per conjugate). ELISA analysis indicated that anti-Ent IgY titers dramatically increased in serum (up to 2,048 fold) and yolk (up to 1,024 fold) of each individual layer upon vaccination with KLH-Ent or CmeC-Ent. However, the BSA-Ent conjugate failed to induce anti-Ent IgY. The hyperimmune egg yolks were pooled and lyophilized for future evaluation of in vivo passive immune protection. Collectively, these studies have provided solid foundation for developing innovative and effective Ent-dependent immune interventions again Gram-negative bacterial infections in the future.
Wang, Huiwen, "Production and Characterization of Enterobactin-specific Antibodies Induced by Novel Enterobactin Conjugate Vaccines. " Master's Thesis, University of Tennessee, 2019.
Portions of this document have been published on the journal Applied and Environmental Microbiology.