Listeriolysin O (LLO) as a Vaccine vehicle

Abstract

Specific Aim: In this project we aimed to test antigen presentation to Listeriolysin O (LLO), the major virulence factor of Listeria monocytogenes (LM). The purpose was to examine the immunogenicity of LLO and its possible use as a carrier of antigens for immunization purposes.ResultsPeptides were made to segments of LLO molecule known to induce CD4 responses. C57Bl/6 mice were immunized with the peptides and their CD4 T cells were isolated, made into hybridomas and used to examine the presentation of LLO ex vivo. The CD4 T cell response was extraordinarily potent when LLO was used as an exogenous antigen testing different Antigen presenting cells (APC). The response to LLO was about 10,000 times more efficient than that for peptide. Looking for a safer molecule without the detrimental effects at higher concentrations, we generated a LLO mutant, the LLOW492A, which has a 90% reduced lytic activity but still maintains the binding capacity to the cell membranes. When we tested this LLO mutant ex vivo the magnitude of the response at low doses of antigen was as strong as that for the wild type LLO. We generated a recombinant LLO protein by attaching a CD4 epitope to the NH2 terminus region. We used a known epitope (45–65 epitope) of the hen egg white lysozyme (HEL) molecule. When we tested the response of a known T cell to 45–65 epitope of HEL linked to the amino terminus region of LLO, the response was about 100 fold stronger than to the peptide alone.Conclusions: We have identified an extraordinarily potent LLO‐specific CD4 T cell response when the whole molecule or a mutant derivative is used as an exogenous antigen. Peptides linked to the protein are likewise highly immunogenic. LLO is a potentially powerful vehicle for immunization.