Doctor of Philosophy / Department of Chemical Engineering / John R. Schlup / Jishu N. Shi / Vaccination is one of the most essential steps in controlling and preventing economically important infectious diseases in livestock. Vaccines need to be effective at producing a high level of immune responses that protect the animal from future encounters with infectious agents. Additional requirements for veterinary vaccines include safety, inexpensive components, and feasibility for large-scale production. These factors make emulsions attractive vaccine adjuvants. The use of emulsions as adjuvants (substances that help to amplify the immune responses to the antigen) has been explored for decades. However, emulsions are commonly produced with expensive and energy-demanding devices which impact the price of the adjuvant, therefore, affecting the price of the vaccines. This study examined low-energy emulsification methods to meet the requirements for a simple and low-cost vaccine manufacture that avoided utilizing complicated equipment. Spontaneous emulsification (SE) and phase inversion composition (PIC) was explored to formulate stable emulsions with nanometer droplet sizes. The study on the impact of oil composition on the formation of emulsions produced by SE revealed that addition of medium-chain triglycerides into the oil phase is beneficial for droplet size reduction and stability of emulsions. Box-Behnken design (BBD) was used to develop mathematical relationships between formulation variables and droplet size, polydispersity, zeta potential, and stability of emulsions formulated via SE. The BBD allowed the study of a simultaneous effect of multiple variables and formulate emulsions with certain physical characteristics, an effect that suggested that there was a more effective approach in designing complex systems like emulsions. New adjuvants containing mixtures of oils and surfactants were developed to produce emulsions with nanoscale droplet diameters and multiple water-in-oil-in-water structures via the PIC approach. The strong antibody responses and the absence of injection site side effects were observed in animals that received emulsion vaccines with experimental adjuvants. Additionally, inexpensive food-grade saponin extract was examined for stabilizing and increasing immunostimulatory activity of oil-in-water emulsion-based adjuvants. The adjuvants demonstrated high immune responses in pigs after co-administration with a subunit protein antigen.

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins that regulate various pathways, particularly those that affect proliferation and cell death. Smac mimetics compounds (SMCs) are IAP antagonists that induce the degradation of two IAPs, cellular IAP 1 (cIAP1) and cellular IAP 2 (cIAP2). cIAP1 and cIAP2 are negative regulators of the alternative NF-κB pathway, which is critical to the regulation, activation, proliferation, and survival of immune cells. Consequently, SMCs can affect immunological responses by providing co-stimulatory signals for antigen-presenting cells or promoting proliferation and activation of T cells. Due to their potent immunomodulatory properties, SMCs are an ideal candidate for new vaccine adjuvants. I sought to demonstrate the potential of SMCs as a vaccine adjuvant by evaluating SMCs effects on dendritic cells (DCs). I demonstrated that SMC treatment of bone marrow derived dendritic cells (BMDCs) induces degradation of both cIAP1 and cIAP2 and leads to activation of the alternative NF-κB signalling pathway. Furthermore, SMC treatment led to upregulation of proteins associated with DC maturation, as well as secretion of pro-inflammatory cytokines. Despite the activating effects elicited by SMCs in vitro, the use of SMCs as an adjuvant for peptide vaccination failed to prevent tumour growth. Further work to determine the best use of SMCs as adjuvants in vivo needs to be done to explore the potential of this class of drugs. Thus, these findings will guide the use of SMCs in adjuvant vaccine therapies for robust protective immunity.

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Toxinas termo-lábeis expressas por Escherichia coli enterotoxigênica (LT-I, LT-IIa e LT-IIb) são adjuvantes sistêmicos e de mucosa. Essas proteínas apresentam reduzida identidade (<14%) em suas subunidades B e ligação a receptores distintos, o que pode resultar em propriedades biológicas diferenciais. O objetivo do trabalho foi avaliar a resposta imune induzida pela toxina LT-IIa e seu pentâmero B (LT-IIaB) administradas pelas vias transcutânea e intradérmica. Inicialmente as proteínas foram expressas em E. coli, purificadas e avaliadas quanto à funcionalidade in vitro. Numa segunda etapa, as propriedades imunomoduladoras de LT-IIa e LT-IIaB, imunogenicidade e atividade adjuvante, foram avaliadas em modelo murino. A resposta imune (humoral e celular) induzida contra ovalbumina (OVA), aplicada como antígeno, foi determinada. Os resultados demonstram que as atividades adjuvantes induzidas por LT-IIa e LT-IIaB variam de acordo com a via de inoculação e que as holotoxinas LT-I e LT-IIa induzem graus de inflamação e ativação de resposta imune distintos em camundongos. / Heat-labile toxins expressed by enterotoxigenic Escherichia coli (LT-I, LT-IIa and LT-IIb) are potent systemic and mucosal adjuvants. These proteins have low identity (<14%) in their B subunits and bind to different receptors, which may result in differential biological properties. The objective of this work was to evaluate the immune response induced by LT-IIa toxin and its pentameric B subunit (LT-IIaB) delivered by transcutaneous and intradermic routes. Initially the proteins of interest were expressed in recombinant E. coli strains, purified and tested for functionality in vitro. In a second moment, the immunomodulatory properties of LT-IIa and LT-IIaB, immunogenicity and adjuvant activity, were evaluated in mouse model. The humoral and cellular immune responses induced against ovalbumin (OVA) used as antigen were determined. The results show that the adjuvant activity of LT-IIa and its B pentamer depends on the route of inoculation and that LT-I and LT-IIa differ both on induction of inflammation and activation of immune responses in mice.

Toxinas termo-lábeis expressas por Escherichia coli enterotoxigênica (LT-I, LT-IIa e LT-IIb) são adjuvantes sistêmicos e de mucosa. Essas proteínas apresentam reduzida identidade (<14%) em suas subunidades B e ligação a receptores distintos, o que pode resultar em propriedades biológicas diferenciais. O objetivo do trabalho foi avaliar a resposta imune induzida pela toxina LT-IIa e seu pentâmero B (LT-IIaB) administradas pelas vias transcutânea e intradérmica. Inicialmente as proteínas foram expressas em E. coli, purificadas e avaliadas quanto à funcionalidade in vitro. Numa segunda etapa, as propriedades imunomoduladoras de LT-IIa e LT-IIaB, imunogenicidade e atividade adjuvante, foram avaliadas em modelo murino. A resposta imune (humoral e celular) induzida contra ovalbumina (OVA), aplicada como antígeno, foi determinada. Os resultados demonstram que as atividades adjuvantes induzidas por LT-IIa e LT-IIaB variam de acordo com a via de inoculação e que as holotoxinas LT-I e LT-IIa induzem graus de inflamação e ativação de resposta imune distintos em camundongos. / Heat-labile toxins expressed by enterotoxigenic Escherichia coli (LT-I, LT-IIa and LT-IIb) are potent systemic and mucosal adjuvants. These proteins have low identity (<14%) in their B subunits and bind to different receptors, which may result in differential biological properties. The objective of this work was to evaluate the immune response induced by LT-IIa toxin and its pentameric B subunit (LT-IIaB) delivered by transcutaneous and intradermic routes. Initially the proteins of interest were expressed in recombinant E. coli strains, purified and tested for functionality in vitro. In a second moment, the immunomodulatory properties of LT-IIa and LT-IIaB, immunogenicity and adjuvant activity, were evaluated in mouse model. The humoral and cellular immune responses induced against ovalbumin (OVA) used as antigen were determined. The results show that the adjuvant activity of LT-IIa and its B pentamer depends on the route of inoculation and that LT-I and LT-IIa differ both on induction of inflammation and activation of immune responses in mice.