<p>Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.</p><p>IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (<italic>Il1r1</italic>-/-) mice given <italic>Il1r1</italic>-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of <italic>Il1r1</italic>-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given <italic>Il1r1</italic>-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.</p> / Dissertation

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.