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Suppressive Oligodeoxynucleotides Inhibit Cytosolic DNA Sensing Pathways: A DissertationThe innate immune system provides an essential first line of defense against infection. Innate immune cells detect pathogens through several classes of Pattern Recognition Receptors (PRR) allowing rapid response to a broad spectrum of infectious agents. Activated receptors initiate signaling cascades that lead to the production of cytokines, chemokines and type I interferons all of which are vital for controlling pathogen load and coordinating the adaptive immune response. Detection of nucleic acids by the innate immune system has emerged as a mechanism by which infection is recognized. Recognition of DNA is complex, influenced by sequence, structure, covalent modification and subcellular localization. Interestingly certain synthetic oligodeoxynucleotides comprised of the TTAGGG motif inhibit proinflammatory responses in a variety of disease models. These suppressive oligodeoxynucleotides (sup ODN) have been shown to directly block TLR9 signaling as well as prevent STAT1 and STAT4 phosphorylation. Recently AIM2 has been shown to engage ASC and assemble an inflammasome complex leading to the caspase-1-dependent maturation of IL-1β and IL-18. The AIM2 inflammasome is activated in response to cytosolic dsDNA and plays an important role in controlling replication of murine cytomegalovirus (MCMV). In the second chapter of this thesis, a novel role for the sup ODN A151 in inhibiting cytosolic nucleic acid sensing pathways is described. Treatment of dendritic cells and macrophages with the A151 abrogated type I IFN, TNF-α and ISG induction in response to cytosolic dsDNA. A151 also reduced INF-β and TNF-α induction in BMDC and BMDM responding to the herpesviruses HSV-1 and MCMV but had no effect on the responses to LPS or Sendai virus. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and MCMV. Although inhibition of interferon-inducing pathways and inflammasome assembly was dependent on backbone composition, sequence differentially affected these pathways. While A151 more potently suppressed the AIM2 inflammasome, a related construct C151, proved to be a more potent inhibitor of interferon induction. A151 suppressed inflammasome signaling by binding to AIM2 and competing with immune-stimulatory DNA. The interaction of A151 and AIM2 prevented recruitment of the adapter ASC and assembly of the macromolecular inflammasome complex. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. The innate immune response to HSV-1 infection is critical for controlling early viral replication and coordinating the adaptive immune response. The cytokines IL-1β and IL-18 are important effector molecules in the innate response to HSV-1 in vivo. However, the PRRs responsible for the production and maturation of these cytokines have not been fully defined. In the third chapter of this thesis, The TLR2-MyD88 pathway is shown to be essential for the induction of pro-IL-1β transcription in dendritic cells and macrophages responding to HSV-1. The HSV-1 immediate-early protein ICP0 has previously been shown to block TLR2 responses and in keeping with this finding, ICP0 blocked pro-IL-1β expression. Following translation, pro-IL-1β exists as an inactive precursor that must be proteolytically cleaved by a multiprotein complex known as the inflammasome to yield its active form. Inflammasomes are composed of cytoplasmic receptors such as NLRP3 or AIM2, the adapter molecule ASC, and pro-caspase-1. In the present study we found that the NLRP3 inflammasome is important for maturation of IL-1β in macrophages and dendritic cells responding to HSV-1. In contrast the related NLRP12 protein controls IL-1β production in neutrophils. These data indicate that sensing of HSV-1 by TLR2 drives pro-IL-1β transcription and infection activates the inflammasome to mature this cytokine. Moreover, these studies reveal cell type-specific roles for NLRP3 and NLRP12 in inflammasome assembly.
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Unique structural solution from a VH3-30 antibody targeting the hemagglutinin stem of influenza A virusesBroadly neutralizing antibodies (bnAbs) targeting conserved influenza A virus (IAV) hemagglutinin (HA) epitopes can provide valuable information for accelerating universal vaccine designs. Here, we report structural details for heterosubtypic recognition of HA from circulating and emerging IAVs by the human antibody 3I14. Somatic hypermutations play a critical role in shaping the HCDR3, which alone and uniquely among VH3-30 derived antibodies, forms contacts with five sub-pockets within the HA-stem hydrophobic groove. 3I14 light-chain interactions are also key for binding HA and contribute a large buried surface area spanning two HA protomers. Comparison of 3I14 to bnAbs from several defined classes provide insights to the bias selection of VH3-30 antibodies and reveals that 3I14 represents a novel structural solution within the VH3-30 repertoire. The structures reported here improve our understanding of cross-group heterosubtypic binding activity, providing the basis for advancing immunogen designs aimed at eliciting a broadly protective response to IAV.
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Respiratory Syncytial Virus (RSV) Induces Innate Immunity through Toll-Like Receptors and Acquired Immunity via the RSV G Protein: A DissertationRespiratory syncytial virus (RSV) causes a common infection that is associated with a range of respiratory illnesses from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children < 1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected as contributing to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs including TLR2, TLR6, TLR3, TLR4, and TLR7 that can potentially interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting TNF-α, IL-6, CCL2 (MCP-1), and CCL5 (RANTES) production. As previously noted, TLR4 also contributed to cytokine activation (71, 90). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation and may play a role in modulating acquired immune responses through DCs. Despite the fact that RSV is the single most important cause of infant upper respiratory tract disease, there are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimera protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). BALB/c mice immunized with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein as good as or better than comparable amounts of UV-inactivated RSV. Furthermore, VLP-H/G induced robust CTL responses in vaccinated animals. Immunization with two or even a single dose of these particles resulted in the complete protection of BALB/c mice from RSV replication in the lungs. Upon RSV challenge of VLP-H/G immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has been previously observed with FI-RSV vaccination. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for RSV.
