We have previously described a model for synthesizing protein- polysaccharide conjugate vaccines which stimulate enhanced antibody responses to both the polysaccharide and the protein components. In this proposal we describe novel approaches to refining this model to produce ~self-immunoadjuvanting~ vaccine constructs that will stimulate enhanced and persistent antibody responses to all of the components in a polysaccharide based vaccine. We have genetically engineered protein-cytokine fusions which will be conjugated to polysaccharides in order to enhance both the T cell dependent response as well as to focus cytokine help onto polysaccharide specific B cells. We have used IL2 and GM-CSF as the cytokine fusion partners, since we have shown that these cytokines can enhance Ig secretion to T cell independent type 2 (TI-type 2) antigens in vitro. As another approach to enhancing the T cell dependent limb of the response, we will construct CD40 ligand (CD40L)-protein fusions which will be conjugated to polysaccharides. We have shown that in vitro CD40L enhances Ig secretion to TI-type 2 antigens in both adult and neonatal cells. We will also genetically engineer a fusion of a cytokine with ~universal~ T cell epitope which will then be conjugated to polysaccharides. Finally, we will conjugate to polysaccharides a B cell stimulating peptide that we have shown induces enhanced B cell proliferation and Ig secretion only in the presence of multivalent mIg crosslinking stimulus. We will immunize normal, neonatal and CD4+ depleted mice via systemic and mucosal routes with these conjugates and measure the titer, persistence and isotype of antibody that is stimulated, as well as the protective efficacy of these antibodies using in vitro and in vivo models. The cellular mechanism underlying the enhancement of antibody responses will also be studied. These combined approaches will lay the groundwork for designing novel polysaccharide-based vaccines that stimulate high titer and persistent antibody in normal, neonatal and immune compromised hosts via systemic or mucosal routes.
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