A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants

PLoS One. 2012;7(9):e45705. doi: 10.1371/journal.pone.0045705. Epub 2012 Sep 24.

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 OVA(257-264) 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-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) 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-OVA(257-264) without adjuvants induced a strong OVA(257-264)-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 necessary for the induction of a cytotoxic response but not for antigen cross presentation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adjuvants, Immunologic / pharmacology
  • Animals
  • Biopolymers
  • CD8-Positive T-Lymphocytes / immunology
  • Cross-Priming / immunology
  • Cytotoxicity, Immunologic / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Flow Cytometry
  • Fluorescent Dyes
  • Lymphocyte Activation
  • Mice
  • Mice, Inbred C57BL
  • Multienzyme Complexes / immunology*
  • Ovalbumin / immunology
  • Polymerase Chain Reaction
  • Toll-Like Receptor 4 / physiology*

Substances

  • Adjuvants, Immunologic
  • Biopolymers
  • Fluorescent Dyes
  • Multienzyme Complexes
  • Toll-Like Receptor 4
  • 6,7-dimethyl-8-ribityllumazine synthase
  • Ovalbumin

Grants and funding

This work was supported in part by grants from the Howard Hughes Medical Institute to F.A.G. (http://www.hhmi.org/grants) and from the Agencia Nacional para la Promoción Científica y Tecnológica ANPCyT (http://www.agencia.gov.ar). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.