Heat shock protein-antigen fusions lose their enhanced immunostimulatory capacity after endotoxin depletion.
Heat shock proteins (HSPs) induce cross-presentation of antigens by dendritic cells (DC) as well as DC maturation. These properties make HSP antigen complexes good candidates to prime CD8 T cell responses against tumor-associated antigens. In this study, we analyzed four different members of the HSP70 family fused to a fragment of ovalbumin (OVA) as a model tumor antigen. E. coli-derived recombinant HSP70-OVA fusion proteins efficiently primed antigen-specific cytotoxic T cells in short-term in vivo immunization assays. Because of concerns that the adjuvant effect of HSPs may be due to endotoxin contamination, we studied this issue in detail. Induction of OVA-specific cytotoxicity was significantly decreased in mice deficient for the LPS receptor, TLR4. After careful removal of endotoxins, immunization with HSP70-OVA failed to prime cytotoxic T cell responses. However, we obtained strong in vivo kill responses when endotoxin-depleted HSP70-OVA was used in combination with the TLR9 ligand CpG oligodeoxynucleotide 1668. Importantly, prophylactic and therapeutic treatment with endotoxin-depleted HSP70-OVA together with CpG significantly delayed the outgrowth of OVA-expressing B16 melanoma cells. However, we were unable to detect significant differences in the magnitudes of immune responses against endotoxin-depleted recombinant OVA vs. endotoxin-depleted HSP70-OVA fusion protein. Thus, immunization with recombinant HSP70-antigen fusion protein does not provide an advantage over recombinant antigen alone when combined with a suitable adjuvant. Altogether, our data suggest that the adjuvant effect of the HSP70 part of the fusion protein is completely lost after endotoxin removal.
Marincek
Boris-Christian
BC
Division of Molecular Immunology (D050), German Cancer Research Center (DKFZ), Heidelberg, Germany.
Kühnle
Marie-Cristine
MC
Srokowski
Cathy
C
Schild
Hansjörg
H
Hämmerling
Günter
G
Momburg
Frank
F
D000276:Adjuvants, Immunologic / Q000378:metabolism*; D000818:Animals; D000941:Antigens / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D045142:Cross-Priming / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004731:Endotoxins / Q000172:deficiency* / Q000276:immunology*; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology*; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009369:Neoplasms / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D010047:Ovalbumin / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000187:drug effects
D000276:Adjuvants, Immunologic; D000941:Antigens; C408982:CPG-oligonucleotide; D004731:Endotoxins; D018840:HSP70 Heat-Shock Proteins; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D010047:Ovalbumin