Nasal immunization with H7 flagellin protects mice against hemolytic uremic syndrome secondary to Escherichia coli O157:H7 gastrointestinal infection.

Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections. Materials and methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo. Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo, and surviving mice presented high titres of anti-H7 and Stx antibodies. Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors.

Make It Simple: (SR-A1+TLR7) Macrophage Targeted NANOarchaeosomes.

Hyperhalophilic archaebacteria exclusively produce sn2,3 diphytanylglycerol diether archaeolipids, unique structures absent in bacteria and eukaryotes. Nanovesicles made of archaeolipids known as nanoarchaeosomes (nanoARC), possess highly stable bilayers, some of them displaying specific targeting ability. Here we hypothesize that nanoARC made from Halorubrum tebenquichense archaebacteria, may constitute efficient carriers for the TLR7 agonist imiquimod (IMQ). NanoARC-IMQ takes advantage of the intense interaction between IMQ and the highly disordered, poorly fluid branched archaeolipid bilayers, rich in archaeol analog of methyl ester of phosphatidylglycerophosphate (PGP-Me), a natural ligand of scavenger receptor A1 (SR-A1). This approach lacks complex manufacture steps required for bilayers labeling, enabling future analytical characterization, batch reproducibility, and adaptation to higher scale production. SR-A1 mediated internalization of particulate material is mostly targeted to macrophages and is extensive because it is not submitted to a negative feedback. A massive and selective intracellular delivery of IMQ may concentrate its effect specifically into the endosomes, where the TLR7 is expressed, magnifying its immunogenicity, at the same time reducing its systemic bioavailability, and therefore it's in vivo adverse effects. NanoARC-IMQ (600-900 nm diameter oligolamellar vesicles of ~-43 mV Z potential) were heavily loaded with IMQ at ~44 µg IMQ/mg phospholipids [~20 folds higher than the non-SR-A1 ligand soyPC liposomes loaded with IMQ (LIPO-IMQ)]. In vitro, nanoARC-IMQ induced higher TNF-α and IL-6 secretion by J774A1 macrophages compared to same dose of IMQ and same lipid dose of LIPO-IMQ. In vivo, 3 subcutaneous doses of nanoARC-IMQ+ 10 µg total leishmania antigens (TLA) at 50 µg IMQ per Balb/C mice, induced more pronounced DTH response, accompanied by a nearly 2 orders higher antigen-specific systemic IgG titers than IMQ+TLA and LIPO-IMQ. The isotype ratio of nanoARC-IMQ+TLA remained ~0.5 indicating, the same as IMQ+TLA, a Th2 biased response distinguished by a pronounced increase in antibody titers, without negative effects on splenocytes lymphoproliferation, with a potential CD8+LT induction 10 days after the last dose. Overall, this first approach showed that highly SR-A1 mediated internalization of heavily loaded nanoARC-IMQ, magnified the effect of IMQ on TLR7 expressing macrophages, leading to a more intense in vivo immune response.

The impact of extracellular acidosis on dendritic cell function.

Dendritic cells (DCs) are the most efficient antigen-presenting cells. They are activated in the periphery by conserved pathogen molecules and by inflammatory mediators produced by a variety of cell types in response to danger signals. It is widely appreciated that inflammatory responses in peripheral tissues are usually associated with the development of acidic microenvironments. Surprisingly, there are relatively few studies directed to analyze the effect of extracellular acidosis on the immune response. We focus on the influence of extracellular acidosis on the function of immature DCs. The results presented here show that acidosis activates DCs. It increases the acquisition of extracellular antigens for MHC class I-restricted presentation and the ability of antigen-pulsed DCs to induce both specific CD8+ CTL and B-cell responses. These findings may have important implications to our understanding of the mechanisms through which DCs sense the presence of infection or inflammation in nonlymphoid tissues.