Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

EGFR-Targeted Granzyme B Expressed in NK Cells Enhances Natural Cytotoxicity and Mediates Specific Killing of Tumor Cells

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells'' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.     

A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.     

Liposome-based Systems for Anti-tumor Vaccination: Influence of Lipopeptide Adjuvants

We have developed liposome-based synthetic constructs incorporating peptide epitope(s) (ErbB2 p63-67 CTL which is overexpressed in many tumors and/or HA 307-319 T-helper) and lipopeptide adjuvants (Pam3CysSerSer, Pam3CysAlaGly) in order to elicit an anti-tumor immune response. The epitopes, derivatized with a linker containing a cysteine residue, were conjugated on preformed vesicles (dia. ～ 100 nm) containing lipopeptides functionalized with thiol reactive groups (maleimide or bromoacetyl). The therapeutic efficacy of these constructs was evaluated on a Balb/c mice tumor model inoculated with syngenic murine renal carcinoma (Renca) cells expressing human ErbB2 (Her2/neu) receptor. A successful therapeutic vaccination was obtained which was antigen specific. Furthermore, it appeared that the nature of the polar head group of the lipopeptide adjuvant and also its type of functionalization influence the efficacy of the construct. In our study, the best results were obtained with formulations containing a Pam₃CSS anchor in association with the CTL and Th epitopes. Considering these promising results studies are in progress with a new generation of liposomes that incorporate a neutral lipid – lacking adjuvant properties – that serves as anchor of the peptide epitopes and new adjuvants synthesized in our laboratory, which are screened for their antitumour activity in a therapeutic setting.     

Middle East Respiratory Syndrome Coronavirus Accessory Protein 4a Is a Type I Interferon Antagonist

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory infection with as yet unclear epidemiology. We previously showed that MERS-CoV counteracts parts of the innate immune response in human bronchiolar cells. Here we analyzed accessory proteins 3, 4a, 4b, and 5 for their abilities to inhibit the type I interferon response. Accessory protein 4a was found to block interferon induction at the level of melanoma differentiation-associated protein 5 (MDA5) activation presumably by direct interaction with double-stranded RNA.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

Harmful Cyanobacterial Blooms: Causes, Consequences, and Controls

Cyanobacteria are the Earth’s oldest oxygenic photoautotrophs and have had major impacts on shaping its biosphere. Their long evolutionary history (～3.5 by) has enabled them to adapt to geochemical and climatic changes, and more recently anthropogenic modifications of aquatic environments, including nutrient over-enrichment (eutrophication), water diversions, withdrawals, and salinization. Many cyanobacterial genera exhibit optimal growth rates and bloom potentials at relatively high water temperatures; hence global warming plays a key role in their expansion and persistence. Bloom-forming cyanobacterial taxa can be harmful from environmental, organismal, and human health perspectives by outcompeting beneficial phytoplankton, depleting oxygen upon bloom senescence, and producing a variety of toxic secondary metabolites (e.g., cyanotoxins). How environmental factors impact cyanotoxin production is the subject of ongoing research, but nutrient (N, P and trace metals) supply rates, light, temperature, oxidative stressors, interactions with other biota (bacteria, viruses and animal grazers), and most likely, the combined effects of these factors are all involved. Accordingly, strategies aimed at controlling and mitigating harmful blooms have focused on manipulating these dynamic factors. The applicability and feasibility of various controls and management approaches is discussed for natural waters and drinking water supplies. Strategies based on physical, chemical, and biological manipulations of specific factors show promise; however, a key underlying approach that should be considered in almost all instances is nutrient (both N and P) input reductions; which have been shown to effectively reduce cyanobacterial biomass, and therefore limit health risks and frequencies of hypoxic events.     

Alexander Böhm (1971–2012)

On November 28, 2012 Alexander (Alex) Böhm, a bacterial geneticist, died at age 41, only a few months after taking up a position as an assistant professor at the LOEWE Center for Synthetic Microbiology in Marburg, Germany. Earlier in 2012 Alex had been diagnosed with an aggressive form of thyroid cancer that left him little time to live his scientific and personal dreams.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.