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1.  Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome 
Immunity  2011;35(2):194-207.
Although Toll-like receptor 9 (TLR9) has been implicated in regulating cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent DNA sensing pathways. Over 6000 ATTTTTAC (“AT-rich”) motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-r motif induce type I IFNs via a pathway that did not involve previously described sensors including TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking both IRF3 and IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.
PMCID: PMC3162998  PMID: 21820332
2.  Cell-type specific recognition of human Metapneumoviruses by RIG-I and TLR7 and viral interference of RIG-I ligand recognition by HMPVB1 Phosphoprotein 
Human Metapneumoviruses (HMPV) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germline encoded pattern recognition receptors and activation of cytokine and type I interferon genes. Recently, the RNA helicase Retinoic acid inducible gene (RIG-I) has been shown to sense HMPV. In this study, we investigated the ability of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I interferons (IFN). Despite the ability of both HMPV-A1 and B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5’ triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells (PDC) displayed a unique ability to sense both the A1 and B1 strains and in this case sensing was via Toll-like receptor (TLR)-7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell-type specific manners.
PMCID: PMC2834787  PMID: 20042593
Viral; Signal Transduction; Knockout mouse
3.  Superior Immunogenicity of Inactivated Whole Virus H5N1 Influenza Vaccine is Primarily Controlled by Toll-like Receptor Signalling 
PLoS Pathogens  2008;4(8):e1000138.
In the case of an influenza pandemic, the current global influenza vaccine production capacity will be unable to meet the demand for billions of vaccine doses. The ongoing threat of an H5N1 pandemic therefore urges the development of highly immunogenic, dose-sparing vaccine formulations. In unprimed individuals, inactivated whole virus (WIV) vaccines are more immunogenic and induce protective antibody responses at a lower antigen dose than other formulations like split virus (SV) or subunit (SU) vaccines. The reason for this discrepancy in immunogenicity is a long-standing enigma. Here, we show that stimulation of Toll-like receptors (TLRs) of the innate immune system, in particular stimulation of TLR7, by H5N1 WIV vaccine is the prime determinant of the greater magnitude and Th1 polarization of the WIV-induced immune response, as compared to SV- or SU-induced responses. This TLR dependency largely explains the relative loss of immunogenicity in SV and SU vaccines. The natural pathogen-associated molecular pattern (PAMP) recognized by TLR7 is viral genomic ssRNA. Processing of whole virus particles into SV or SU vaccines destroys the integrity of the viral particle and leaves the viral RNA prone to degradation or involves its active removal. Our results show for a classic vaccine that the acquired immune response evoked by vaccination can be enhanced and steered by the innate immune system, which is triggered by interaction of an intrinsic vaccine component with a pattern recognition receptor (PRR). The insights presented here may be used to further improve the immune-stimulatory and dose-sparing properties of classic influenza vaccine formulations such as WIV, and will facilitate the development of new, even more powerful vaccines to face the next influenza pandemic.
Author Summary
The rise and spread of the highly pathogenic avian H5N1 influenza virus has seriously increased the risk of a new influenza pandemic. However, the number of vaccine doses that can be produced with today's production capacity will fall short of the demand in times of a pandemic. Use of inactivated whole virus (WIV) vaccines, which are more immunogenic than split virus or subunit vaccines in an unprimed population, could contribute to a dose-sparing strategy. Yet, the mechanisms underlying the superior immunogenicity of WIV vaccine formulations are unknown. Here, we demonstrate that the viral RNA present in inactivated virus particles is crucial for the improved immunogenic properties of WIV in mice. By triggering Toll-like receptor 7 (TLR7), the viral RNA activates innate immune mechanisms that augment and determine subsequent adaptive responses. Efficient TLR7 signalling is lost in split virus and subunit vaccines with the processing steps that lead to disruption of the integrity of the virus particle and exclusion of the RNA. Our results prove for the first time to our knowledge that the immune-potentiating mechanism of a classic vaccine is based on activation of the innate immune system by one of its structural components. These findings may reflect a general principle for viral vaccines and provide a rational basis for further improvement of influenza vaccines, which are urgently needed in the face of the current H5N1 pandemic threat.
PMCID: PMC2516931  PMID: 18769719
4.  The chemotherapeutic agent DMXAA potently and specifically activates the TBK1–IRF-3 signaling axis 
The Journal of Experimental Medicine  2007;204(7):1559-1569.
Vascular disrupting agents (VDAs) represent a novel approach to the treatment of cancer, resulting in the collapse of tumor vasculature and tumor death. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a VDA currently in advanced phase II clinical trials, yet its precise mechanism of action is unknown despite extensive preclinical and clinical investigations. Our data demonstrate that DMXAA is a novel and specific activator of the TANK-binding kinase 1 (TBK1)–interferon (IFN) regulatory factor 3 (IRF-3) signaling pathway. DMXAA treatment of primary mouse macrophages resulted in robust IRF-3 activation and ∼750-fold increase in IFN-β mRNA, and in contrast to the potent Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), signaling was independent of mitogen-activated protein kinase (MAPK) activation and elicited minimal nuclear factor κB–dependent gene expression. DMXAA-induced signaling was critically dependent on the IRF-3 kinase, TBK1, and IRF-3 but was myeloid differentiation factor 88–, Toll–interleukin 1 receptor domain–containing adaptor inducing IFN-β–, IFN promoter-stimulator 1–, and inhibitor of κB kinase–independent, thus excluding all known TLRs and cytosolic helicase receptors. DMXAA pretreatment of mouse macrophages induced a state of tolerance to LPS and vice versa. In contrast to LPS stimulation, DMXAA-induced IRF-3 dimerization and IFN-β expression were inhibited by salicylic acid. These findings detail a novel pathway for TBK1-mediated IRF-3 activation and provide new insights into the mechanism of this new class of chemotherapeutic drugs.
PMCID: PMC2118649  PMID: 17562815

Results 1-4 (4)