The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.

Diabetes mellitus is presently recognized as the sixth leading cause of death in the United States, affecting over 20 million people. Diabetes is a condition characterized by high blood glucose due to an insulin deficiency or resistance. Type I, which comprises 5-10% of all cases, results from the destruction of pancreatic beta cells in the islets of Langerhans. The current treatment for type I diabetes is insulin administration through injection or pump. Purification, production, and storage of this insulin proves to be quite costly. By producing biologically functional insulin with oral delivery capabilities through chloroplast genetic engineering, many of these costs could be cut back. In addition, the possibility of providing the C-peptide currently lacking in commercially available insulin becomes available. The Daniell lab inserted cholera toxin B-subunit (CTB) fused proinsulin, containing three furin cleavage sites, (CTB-pins Fx3) into the tobacco chloroplasts of Petit Havana via particle gun bombardment. The insertion of three furin cleavage sites along with fusion to CTB will enable the insulin to sustain function when orally administered. Transgenic plants from the second generation were then analyzed and tested for quantification of the CTB-pins Fx3 gene. Tobacco leaves of varying ages were compared to determine expression levels. The resulting data is pertinent for future production of both orally deliverable insulin and more cost effective injectable insulin.