A Polymeric Protein Induces Specific Cytotoxicity in a TLR4 Dependent Manner in the Absence of Adjuvants

Abstract

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA257-264 and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA257-264 in vivo. BLS-OVA257-264 immunization induced the proliferation of OVA257-264-specific CD8+ lymphocytes and also increased the percentage of OVA257-264-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA257-264-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA257-264 induced the generation of CD8+ effector cells. BLS-OVA257-264 was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA257-264 without adjuvants induced a strong OVA257-264-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor.These data show that TLR4 signaling is necesary for the induction of a cytotoxic response but not for antigen cross presentation.