ABSTRACT
Enterotoxin-based adjuvants including cholera toxin and heat-labile toxin (LT) are powerful manipulators of mucosal immunity; however, past clinical trials identified unacceptable neurological toxicity when LT or mutant AB5 adjuvant proteins were added to intranasal vaccines. Here, we examined the isolated enzymatic A1 domain of LT (LTA1) for intranasal safety and efficacy in combination with influenza (flu) vaccination. LTA1-treated mice exhibited no neurotoxicity, as measured by olfactory system testing and H&E staining of nasal tissue in contrast with cholera toxin. In vaccination studies, intranasal LTA1 enhanced immune responses to inactivated virus antigen and subsequent protection against H1N1 flu challenge in mice (8-week or 24-months). In addition, lung H1N1 viral titers post-challenge correlated to serum antibody responses; however, enhanced protection was also observed in µMT mice lacking B-cells while activation and recruitment of CD4 T-cells into the lung was apparent. Thus, we report that LTA1 protein is a novel, safe and effective enterotoxin adjuvant that improves protection of an intranasal flu vaccination by a mechanism that does not appear to require B-cells.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Aging/immunology , B-Lymphocytes/immunology , Enterotoxins/administration & dosage , Lymphocyte Depletion , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/prevention & control , Adjuvants, Immunologic/pharmacology , Administration, Intranasal , Animals , Antibodies/blood , Antibody Formation/drug effects , B-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Dose-Response Relationship, Immunologic , Enterotoxins/immunology , Enterotoxins/toxicity , Female , Immunity, Mucosal/drug effects , Immunization , Inflammation/pathology , Influenza A Virus, H1N1 Subtype/immunology , Lung/virology , Lymphocyte Activation/drug effects , Mast Cells/drug effects , Mast Cells/pathology , Mice, Inbred C57BL , Orthomyxoviridae Infections/blood , Orthomyxoviridae Infections/virologyABSTRACT
5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.
