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The overall goal of this research is to develop more effective formulations of aluminum adjuvanted vaccines. <P>The specific objectives of this research are: <br/>1.Optimize the interaction between antigens and aluminum adjuvant particles by modifying the antigens with a linker agent terminal phosphate groups. Terminal phosphate groups can exchange with hydroxyl groups at the surface of the aluminum adjuvants resulting in a stable bond. By varying the number of linkers and the length of the linkers, the interaction between antigen and adjuvant can be changed and optimized. <br/>2.Determine the kinetics of cell infiltration and gene expression at the injection site following intramuscular injection of the aluminum-adjuvanted vaccines. <br/>3.Determine the effect of manipulation of the inflammatory response at the injection site on the subsequent immune response. Selective depletion of types of inflammatory cells and blocking of specific cytokines may result in a qualitatively and quantitatively enhanced immune response. Information obtained through these studies will be published in peer-reviewed journals, presented at national and international meetings and may generate commercial products.
Non-Technical Summary:<br/>
Aluminum adjuvants are widely used in veterinary and human vaccines. The proposed research is aimed at two approaches to increase the immune response to aluminum adjuvanted vaccines. The first is to modify the interaction between aluminum adjuvants and antigens by adding phosphate groups to the vaccine antigens. This would lead to prolonged retention of antigen at the injection site. The second approach is to change the type of inflammation at the injection site. The inflammation is an important determinant of the immune response that develops. This research may result in new vaccines and in improvements of existing vaccines by dose sparing of vaccine antigens and a reduction in the number of doses needed to induce a protective immune response.
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Approach:<br/>
Objective 1. Linkers will be prepared that vary in length and composition, and the number of linkers per antigen molecule will be varied. This work will initially be carried out with two model antigens, hen egg lysozyme (HEL) and a genetically modified inactive form of diphtheria toxin, CRM197. Other vaccine antigens may be included as they become available. The interaction between adjuvants and modified antigens will be determined in vitro by measuring the adsorption. The effect of the linker modification on the antibody response will be determined in a mouse model. Geometric mean titers will be calculated for each group. A one-way ANOVA will be performed on log2-transformed titers to determine if there are significant differences between groups. If a significant difference is detected at P < 0.05, a Bonferroni multiple comparison post-hoc analysis will be performed to compare the differences between groups.
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Objective 2.A model vaccine composed of aluminum hydroxide adjuvant with ovalbumin will be prepared. The vaccine will be injected into the gastrocnemius muscle of mice. Injection sites will be collected 6 hours, 1 day, 2 days, and 7 days after injection. In some experiments, the mice will first be immunized intraperitoneally with ovalbumin or sterile saline (as a control) followed 3 weeks later by intramuscular injection of the aluminum-adjuvanted vaccines. The kinetics and type of inflammation at the injection site in naive and previously immunized mice will be compared by immunofluorescence and RT-PCR. The significance of differences in histology scores between groups will be determined by nonparametric analysis of variance (Kruskall Wallis) followed by Dunn's multiple comparison test. Differences will be considered significant at P < 0.05. For mRNA expression the geometric means of fold increases in mRNA expression will be compared on log-2 transformed data by one-way ANOVA followed by a Bonferroni multiple comparison post-hoc analysis.
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Objective 3. Monoclonal antibody-based depletion and genetically engineered mice will be used to determine the role of specific cell types. The target cell type will depend on the results of the experiments in Objective 2.