The intranasal route of vaccination has become the predominant one used m mucosal immunology in order to induce both systemic and mucosal T cell and antibody (Ab) responses. Nasal application of vaccines with adjuvants is easy to perform, requires l0 - 20 fold less material than does oral immunization and the vaccine and adjuvant are not subjected to degradative enzymes as they are in the GI tract. Despite these advantages, it is often not appreciated that the nasal region is extensively innervated by the olfactory bulb, which is directly connected via olfactory nerves to the olfactory epithelium. Both native enterotoxins like cholera and E. coli labile toxin (CT and LT), and nontoxic derivatives have pentameric B subunits, which bind to GM1 (CT) and to GM2 and asialo GM1 (LT) which are expressed by the olfactory epithelium and the associated olfactory nerves. It was shown that either CT or anti-GM1 Abs induced CNS lesions when injected into the lumbosacral subarachnoid space. This toxicity is consistent with expression of GM1 by astroglia and neurons of the CNS and by microglial cells of the cerebellar cortex. We have therefore postulated that intranasal application of nCT or nontoxic mCTs, which have intact (pentameric) CT-B, could enter the CNS through GM1 binding to olfactory epithelium and nerves with subsequent transport into the olfactory bulb. This would postulate that retrograde axonal transport would bypass the blood / brain barrier. Further, either nCT or mCT could potentially damage the CNS and even induce CNS uptake of co-nasally delivered vaccine proteins. The first Specific Aim to test this hypothesis will actually examine the trafficking of nCT or mCTs in olfactory epithelium and the CNS and whether these molecules induce neuropathologic changes. The second Specific Aim will determine if nCT or mCTs also induce uptake and trafficking of co-administered vaccine proteins into the nasal olfactory epithelium, nerves, and bulb. The third Specific Aim will determine potential toxicity of nCT and mCTs for cultured astrocytes and microglial cells, including inflammatory cytokine production. The fourth Specific Aim will extend this analysis of toxic, inflammatory and apoptotic responses to the nerve cells themselves in the olfactory bulb when exposed to nCT or mCTs. The final Specific Aim will assess long-term effects of CT-B binding to GM1 on neural cells, including the induction of autoimmune anti-GM1 Abs. These proposed studies will provide essential new information to determine potential side effects of CT-B derivatives as nasal adjuvants, and point to ways where these toxic effects can be avoided.
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