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* Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, Yokohama, Japan;
Department of Molecular Protozoology and
Host Defense, Research Institute for Microbial Diseases, Osaka University, Suita, Japan;
World Premier International Immunology Frontier Research Center, Osaka University, Suita, Japan;
¶ Laboratory for Cell Function Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, Wako, Japan;
|| Kihara Institute for Biological Research, Yokohama City University Graduate School of Integrated Science, Totsuka, Japan; and
# Department of Bioregulation, National Institute of Infectious Diseases, Higashimurayama-shi, Japan
Modulation of intracellular signaling using cell-permeable polypeptides is a promising technology for future clinical applications. To develop a novel approach to activate innate immune signaling by synthetic polypeptides, we characterized several different polypeptides derived from the caspase recruitment domain (CARD) of IFN-β promoter stimulator 1, each of which localizes to a different subcellular compartment. Of particular interest was, N'-CARD, which consisted of the nuclear localization signal of histone H2B and the IFN-β promoter stimulator 1CARD and which localized to the nucleus. This polypeptide led to a strong production of type I IFNs and molecular and genetic analyses showed that nuclear DNA helicase II is critically involved in this response. N'-CARD polypeptide fused to a protein transduction domain (N'-CARD-PTD) readily transmigrated from the outside to the inside of the cell and triggered innate immune signaling. Administration of N'-CARD-PTD polypeptide elicited production of type I IFNs, maturation of bone marrow-derived dendritic cells, and promotion of vaccine immunogenicity by enhancing Ag-specific Th1-type immune responses, thereby protecting mice from lethal influenza infection and from outgrowth of transplanted tumors in vivo. Thus, our results indicate that the N'-CARD-PTD polypeptide belongs to a new class of vaccine adjuvant that directly triggers intracellular signal transduction by a distinct mechanism from those engaged by conventional vaccine adjuvants, such as TLR ligands.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported, in part, by the Strategic Research Project of Yokohama City University (K18022 to F.T.), the Advancement of Medical Sciences from Yokohama Medical Foundation (to F.T. and K.K.), the National Institute of Biomedical Innovation (to K.O.), the Yasuda Medical Foundation (to F.T.), the Uehara Memorial Foundation (to F.T.), and a Grant-in-aid for Scientific Research (20590477 to F.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
2 Address correspondence and reprint requests to Dr. Fumihiko Takeshita, Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama, Japan. E-mail address: takesita{at}yokohama-cu.ac.jp
3 K.K. and F.T. contributed equally to this work.
4 Abbreviations used in this paper: RLH, RIG-I-like helicase; B-DNA, B-form DNA; IPS-1, IFN-β promoter stimulator 1; TRAF, TNFR-associating factor; TANK, TRAF family member-associated NF-
B activator; TBK1, TANK binding kinase 1; IKK, IB kinase; IKKi, inducible IKK; IRF3, IFN regulatory factor 3; CARD, caspase recruitment domain; N'-CARD, fusion of the NH2-terminal nuclear localization signal of histone H2B to the IPS-1 CARD; PTD, protein transduction domain; TMD, transmembrane domain; NLS, nuclear localization signal; NDH, nuclear DNA helicase II; ODN, oligodeoxynucleotide; flu vax, influenza split-product vaccine; DC, dendritic cell; FL, full length; BM-DC, bone marrow-derived dendritic cell.
5 The online version of this article contains supplemental information.
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