Efficacious Recombinant Influenza Vaccines Produced by High Yield Bacterial Expression: A Solution to Global Pandemic and Seasonal Needs

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines

Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation

Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE2) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20–30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation

To respond or not to respond - a personal perspective of intestinal tolerance

For many years, the intestine was one of the poor relations of the immunology world, being a realm inhabited mostly by specialists and those interested in unusual phenomena. However, this has changed dramatically in recent years with the realization of how important the microbiota is in shaping immune function throughout the body, and almost every major immunology institution now includes the intestine as an area of interest. One of the most important aspects of the intestinal immune system is how it discriminates carefully between harmless and harmful antigens, in particular, its ability to generate active tolerance to materials such as commensal bacteria and food proteins. This phenomenon has been recognized for more than 100 years, and it is essential for preventing inflammatory disease in the intestine, but its basis remains enigmatic. Here, I discuss the progress that has been made in understanding oral tolerance during my 40 years in the field and highlight the topics that will be the focus of future research

Adjuvant TACE inhibitor treatment improves the outcome of TLR2(-/- )mice with experimental pneumococcal meningitis

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 10(3 )CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2(-/-), CD14(-/- )and CD14(-/-)/TLR2(-/- )mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2(-/- )mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2(-/- )than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14(-/- )and TLR2(-/-)/CD14(-/- )mice, but only 79% of TLR2(-/- )mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2(-/- )and CD14(-/- )mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2(-/- )mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis

MFG-E8 Regulates the Immunogenic Potential of Dendritic Cells Primed with Necrotic Cell-Mediated Inflammatory Signals

Dendritic cells (DC) manipulate tissue homeostasis by recognizing dying cells and controlling immune functions. However, the precise mechanisms by which DC recognize different types of dying cells and devise distinct immunologic consequences remain largely obscure. Herein, we demonstrate that Milk-fat globule-EGF VIII (MFG-E8) is a critical mediator controlling DC immunogenicity in inflammatory microenvironments. MFG-E8 restrains DC-mediated uptake and recognition of necrotic cells. The MFG-E8-mediated suppression of necrotic cell uptake by DC resulted in the decreased proinflammatory cytokines production and activated signal components such as STAT3 and A20, which are critical to maintain tolerogenic properties of DC. Furthermore, the DC-derived MFG-E8 negatively regulates the cross-priming and effector functions of antigen-specific T cells upon recognition of necrotic cells. MFG-E8 deficiency enhances an ability of necrotic cell-primed DC to stimulate antitumor immune responses against established tumors. Our findings define what we believe to a novel mechanism whereby MFG-E8 regulates the immunogenicity of DC by modulating the modes of recognition of dying cells. Manipulating MFG-E8 levels in DC may serve as a useful strategy for controlling inflammatory microenvironments caused by various pathological conditions including cancer and autoimmunity

Endometrial cells sense and react to tissue damage during infection of the bovine endometrium via interleukin 1

Cells generate inflammatory responses to bacteria when pattern recognition receptors bind pathogen-associated molecules such as lipopolysaccharide. Cells may also respond to tissue damage by sensing damage-associated molecules. Postpartum bacterial infections of the bovine uterus cause endometritis but the risk of disease is increased by tissue trauma triggered by dystocia. Animals that suffered dystocia had increased concentrations of inflammatory mediators IL-8, IL-1β and IL-1α in vaginal mucus 3 weeks postpartum, but they also had more bacteria than normal animals. Ex vivo organ cultures of endometrium, endometrial cells and peripheral blood monocytes did not generate inflammatory responses to prototypical damage molecules, HMGB1 or hyaluronan, or to necrotic cells; although they secreted IL-6 and IL-8 in a concentration-dependent manner when treated with IL-1α. However, necrotic endometrial cells did not accumulate intracellular IL-1α or release IL-1α, except when pre-treated with lipopolysaccharide or bacteria. Endometrial cell inflammatory responses to IL-1α were dependent on the cognate receptor IL-1R1, and the receptor adaptor protein MyD88, and the inflammatory response to IL-1α was independent of the response to lipopolysaccharide. Rather than a typical damage-associated molecule, IL-1α acts to scale the inflammatory response in recognition that there is a combination of pathogen challenge followed by endometrial cell damage

MHC Class I Endosomal and Lysosomal Trafficking Coincides with Exogenous Antigen Loading in Dendritic Cells

BACKGROUND: Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing. CONCLUSIONS/SIGNIFICANCE: We conclude that DCs have 'hijacked' and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place

Sp1 Is Essential for p16(INK4a) Expression in Human Diploid Fibroblasts during Senescence

BACKGROUND: p16 (INK4a) tumor suppressor protein has been widely proposed to mediate entrance of the cells into the senescent stage. Promoter of p16 (INK4a) gene contains at least five putative GC boxes, named GC-I to V, respectively. Our previous data showed that a potential Sp1 binding site, within the promoter region from −466 to −451, acts as a positive transcription regulatory element. These results led us to examine how Sp1 and/or Sp3 act on these GC boxes during aging in cultured human diploid fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: Mutagenesis studies revealed that GC-I, II and IV, especially GC-II, are essential for p16 (INK4a) gene expression in senescent cells. Electrophoretic mobility shift assays (EMSA) and ChIP assays demonstrated that both Sp1 and Sp3 bind to these elements and the binding activity is enhanced in senescent cells. Ectopic overexpression of Sp1, but not Sp3, induced the transcription of p16 (INK4a). Both Sp1 RNAi and Mithramycin, a DNA intercalating agent that interferes with Sp1 and Sp3 binding activities, reduced p16 (INK4a) gene expression. In addition, the enhanced binding of Sp1 to p16 (INK4a) promoter during cellular senescence appeared to be the result of increased Sp1 binding affinity, not an alteration in Sp1 protein level. CONCLUSIONS/SIGNIFICANCE: All these results suggest that GC- II is the key site for Sp1 binding and increase of Sp1 binding activity rather than protein levels contributes to the induction of p16 (INK4a) expression during cell aging