• Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

      Ullah, Sami; Seidel, Katja; Türkkan, Sibel; Warwas, Dawid Peter; Dubich, Tatyana; Rohde, Manfred; Hauser, Hansjörg; Behrens, Peter; Kirschning, Andreas; Köster, Mario; et al. (2018-12-23)
      Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release.
    • Hepatocyte-specific suppression of microRNA-221-3p mitigates liver fibrosis.

      Tsay, Hsin-Chieh; Yuan, Qinggong; Balakrishnan, Asha; Kaiser, Marina; Möbus, Selina; Kozdrowska, Emilia; Farid, Marwa; Tegtmeyer, Pia-Katharina; Borst, Katharina; Vondran, Florian W R; et al. (Elsevier, 2018-12-22)
      Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The crosstalk between hepatocytes and hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis. Although ample efforts have been devoted to elucidate the functions of HSCs during liver fibrosis, the regulatory functions of hepatocytes remain elusive. Using an unbiased functional microRNA (miRNA) screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via miRNA modulation. The in vivo functional analyses were performed by inhibiting miRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride- and 3,5-di-diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. Blocking miRNA-221-3p function in hepatocytes during chronic liver injury facilitated recovery of the liver and faster resolution of the deposited extracellular matrix. Furthermore, we demonstrate that reduced secretion of C-C motif chemokine ligand 2, as a result of post-transcriptional regulation of GNAI2 (G protein alpha inhibiting activity polypeptide 2) by miRNA-221-3p, mitigates liver fibrosis. Collectively, miRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis.
    • Developmental induction of human T-cell responses against Candida albicans and Aspergillus fumigatus.

      Vogel, Katrin; Pierau, Mandy; Arra, Aditya; Lampe, Karen; Schlueter, Dirk; Arens, Christoph; Brunner-Weinzierl, Monika C; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2018-11-15)
      The origin of human T-cell responses against fungal pathogens early in life is not clearly understood. Here, we show that antifungal T-cell responses are vigorously initiated within the first years of life against lysates and peptides of Candida albicans or Aspergillus fumigatus, presented by autologous monocytes. The neonatal responding T-cell pool consists of 20 different TCR-V
    • Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants.

      Khera, Tanvi; Behrendt, Patrick; Bankwitz, Dorothea; Brown, Richard J P; Todt, Daniel; Doepke, Mandy; Ghafoor Khan, Abdul; Schulze, Kai; Law, John; Logan, Michael; et al. (Elsevier, 2018-11-12)
      Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an HCV vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. We created HCV variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies.
    • Importance of flagella in acute and chronic Pseudomonas aeruginosa infections.

      Lorenz, Anne; Preuße, Matthias; Bruchmann, Sebastian; Pawar, Vinay; Grahl, Nora; Pils, Marina C; Nolan, Laura M; Filloux, Alain; Weiss, Siegfried; Häussler, Susanne; et al. (Wiley-Blackwell, 2018-11-08)
      Pseudomonas aeruginosa is an environmental microorganism and a causative agent of diverse acute and chronic, biofilm-associated infections. Advancing research-based knowledge on its adaptation to conditions within the human host is bound to reveal novel strategies and targets for therapeutic intervention. Here, we investigated the traits that P. aeruginosa PA14 as well as a virulence attenuated ΔlasR mutant need to survive in selected murine infection models. Experimentally, the genetic programs that the bacteria use to adapt to biofilm-associated versus acute infections were dissected by passaging transposon mutant libraries through mouse lungs (acute) or mouse tumours (biofilm-infection). Adaptive metabolic changes of P. aeruginosa were generally required during both infection processes. Counter-selection against flagella expression was observed during acute lung infections. Obviously, avoidance of flagella-mediated activation of host immunity is advantageous for the wildtype bacteria. For the ΔlasR mutant, loss of flagella did not confer a selective advantage. Apparently, other pathogenesis mechanisms are active in this virulence attenuated strain. In contrast, the infective process of P. aeruginosa in the chronic biofilm model apparently required expression of flagellin. Together, our findings imply that the host immune reactions against the infectious agent are very decisive for acuteness and duration of the infectious disease. They direct disease outcome.
    • Breaking the vicious cycle of antibiotic killing and regrowth of biofilm-residing .

      Müsken, Mathias; Pawar, Vinay; Schwebs, Timo; Bähre, Heike; Felgner, Sebastian; Weiss, Siegfried; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-10-08)
      Biofilm-residing bacteria embedded in an extracellular matrix are protected from diverse physico-chemical insults. In addition to the general recalcitrance of biofilm-bacteria, high bacterial loads in biofilm-associated infections significantly diminishes the efficacy of antimicrobials due to a low per-cell antibiotic concentration. Accordingly, present antimicrobial treatment protocols, that have been established to serve the eradication of acute infections, fail to clear biofilm-associated chronic infections. In the present study, we applied automated confocal microscopy on Pseudomonas aeruginosa to monitor dynamic killing of biofilm-grown bacteria by tobramycin and colistin in real-time. We revealed that the time required for surviving bacteria to repopulate the biofilm could be taken as measure for effectiveness of the antimicrobial treatment. It depends on the: i) nature and concentration of the antibiotic, ii) duration of antibiotic treatment; iii) application as mono or combination therapy and iv) time intervals of drug administration. The vicious cycle of killing and repopulation of biofilm bacteria could also be broken in an in vivo model system by applying successive antibiotic dosages with time intervals that do not allow full reconstitution of the biofilm communities. Treatment regimens that consider the important aspects of antimicrobial killing kinetics bear the potential to improve control of biofilm regrowth. This is an important and underestimated factor that is bound to ensure sustainable treatment success of chronic infections.
    • Heparin: role in protein purification and substitution with animal-component free material.

      Bolten, Svenja Nicolin; Rinas, Ursula; Scheper, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2018-10-01)
      Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.
    • Itaconic acid indicates cellular but not systemic immune system activation.

      Meiser, Johannes; Kraemer, Lisa; Jaeger, Christian; Madry, Henning; Link, Andreas; Lepper, Philipp M; Hiller, Karsten; Schneider, Jochen G; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-08-14)
      Itaconic acid is produced by mammalian leukocytes upon pro-inflammatory activation. It appears to inhibit bacterial growth and to rewire the metabolism of the host cell by inhibiting succinate dehydrogenase. Yet, it is unknown whether itaconic acid acts only intracellularly, locally in a paracrine fashion, or whether it is even secreted from the inflammatory cells at meaningful levels in peripheral blood of patients with severe inflammation or sepsis. The aim of this study was to determine the release rate of itaconic acid from pro-inflammatory activated macrophages
    • Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.

      Roth, Gustavo; Vanz, Ana Letícia; Lünsdorf, Heinrich; Nimtz, Manfred; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-09)
      Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.
    • Chronic hepatitis C virus infection irreversibly impacts human natural killer cell repertoire diversity.

      Strunz, Benedikt; Hengst, Julia; Deterding, Katja; Manns, Michael P; Cornberg, Markus; Ljunggren, Hans-Gustaf; Wedemeyer, Heiner; Björkström, Niklas K; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-11)
      Diversity is a central requirement for the immune system's capacity to adequately clear a variety of different infections. As such, natural killer (NK) cells represent a highly diverse population of innate lymphocytes important in the early response against viruses. Yet, the extent to which a chronic pathogen affects NK cell diversity is largely unknown. Here we study NK cell functional diversification in chronic hepatitis C virus (HCV) infection. High-dimensional flow cytometer assays combined with stochastic neighbor embedding analysis reveal that chronic HCV infection induces functional imprinting on human NK cells that is largely irreversible and persists long after successful interventional clearance of the virus. Furthermore, HCV infection increases inter-individual, but decreases intra-individual, NK cell diversity. Taken together, our results provide insights into how the history of infections affects human NK cell diversity.
    • RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B.

      Broglia, Laura; Materne, Solange; Lécrivain, Anne-Laure; Hahnke, Karin; Le Rhun, Anaïs; Charpentier, Emmanuelle; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5' untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5' UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5' UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5' UTR and on the role of RNase Y in speB regulation.
    • Induced B Cell Development in Adult Mice.

      Brennecke, Anne-Margarete; Düber, Sandra; Roy, Bishnudeo; Thomsen, Irene; Garbe, Annette I; Klawonn, Frank; Pabst, Oliver; Kretschmer, Karsten; Weiss, Siegfried; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
      We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.
    • Budesonide in Autoimmune Hepatitis: The Right Drug at the Right Time for the Right Patient.

      Manns, Michael P; Jaeckel, Elmar; Taubert, Richard; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-11-08)
    • Hepatitis D virus in Africa: several unmet needs.

      Manns, Michael P; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-10)
    • Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

      Gödecke, Natascha; Zha, Lisha; Spencer, Shawal; Behme, Sara; Riemer, Pamela; Rehli, Michael; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, D38124 Braunschweig, Germany. (2017-09-19)
      Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.
    • Linoleic and palmitoleic acid block streptokinase-mediated plasminogen activation and reduce severity of invasive group A streptococcal infection

      Rox, Katharina; Jansen, Rolf; Loof, Torsten G.; Gillen, Christine M.; Bernecker, Steffen; Walker, Mark J.; Chhatwal, Gursharan Singh; Müller, Rolf; Helmholtz-Institut für pharmazeutische Forschung Saarland,Universitätscampus E8.1, 66123 Saarbrücken, Germany. (2017-09-18)
      In contrast to mild infections of Group A Streptococcus (GAS) invasive infections of GAS still pose a serious health hazard: GAS disseminates from sterile sites into the blood stream or deep tissues and causes sepsis or necrotizing fasciitis. In this case antibiotics do not provide an effective cure as the bacteria are capable to hide from them very quickly. Therefore, new remedies are urgently needed. Starting from a myxobacterial natural products screening campaign, we identified two fatty acids isolated from myxobacteria, linoleic and palmitoleic acid, specifically blocking streptokinase-mediated activation of plasminogen and thereby preventing streptococci from hijacking the host’s plasminogen/plasmin system. This activity is not inherited by other fatty acids such as oleic acid and is not attributable to the killing of streptococci. Moreover, both fatty acids are superior in their inhibitory properties compared to two clinically used drugs (tranexamic or ε-amino caproic acid) as they show 500–1000 fold lower IC50 values. Using a humanized plasminogen mouse model mimicking the clinical situation of a local GAS infection that becomes systemic, we demonstrate that these fatty acids ameliorate invasive GAS infection significantly. Consequently, linoleic and palmitoleic acid are possible new options to combat GAS invasive diseases.
    • Analysis of mitochondrial metabolism in situ: Combining stable isotope labeling with selective permeabilization.

      Nonnenmacher, Yannic; Palorini, Roberta; d'Herouël, Aymeric Fouquier; Krämer, Lisa; Neumann-Schaal, Meina; Chiaradonna, Ferdinando; Skupin, Alexander; Wegner, Andre; Hiller, Karsten; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09)
      To date, it is well-established that mitochondrial dysfunction does not only play a vital role in cancer but also in other pathological conditions such as neurodegenerative diseases and inflammation. An important tool for the analysis of cellular metabolism is the application of stable isotope labeled substrates, which allow for the tracing of atoms throughout metabolic networks. While such analyses yield very detailed information about intracellular fluxes, the determination of compartment specific fluxes is far more challenging. Most approaches for the deconvolution of compartmented metabolism use computational models whereas experimental methods are rare. Here, we developed an experimental setup based on selective permeabilization of the cytosolic membrane that allows for the administration of stable isotope labeled substrates directly to mitochondria. We demonstrate how this approach can be used to infer metabolic changes in mitochondria induced by either chemical or genetic perturbations and give an outlook on its potential applications.
    • Sonderforschungsbereich SFB 738: Optimierung konventioneller und innovativer Transplantate

      Manns, Michael P; Huber, Petra; Jaeckel, Elmar; Helmholtz Zentrum für Infektionsforschung GmbH, Inhoofenstr. 7, 38124 Braunschweig, Germany. (2017-08-09)
    • Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions.

      Swierzy, Izabela J; Händel, Ulrike; Kaever, Alexander; Jarek, Michael; Scharfe, Maren; Schlüter, Dirk; Lüder, Carsten G K; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-03)
      The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.
    • Human Anti-Lipopolysaccharid (LPS) antibodies against Legionella with high species specificity.

      Kuhn, Philipp; Thiem, Stefanie; Steinert, Michael; Purvis, Duncan; Lugmayr, Veronika; Treutlein, Ulrich; Plobner, Lutz; Leiser, Robert-Matthias; Hust, Michael; Dübel, Stefan; et al. (2017-07-19)
      Legionella are Gram-negative bacteria that are ubiquitously present in natural and man-made water reservoirs. When humans inhale aerosolized water contaminated with Legionella, alveolar macrophages can be infected, which may lead to a life-threatening pneumonia called Legionnaires' disease. Due to the universal distribution of Legionella in water and their potential threat to human health, the Legionella concentration in water for human use must be strictly monitored, which is difficult since the standard detection still relies on lengthy cultivation and analysis of bacterial morphology. In this study, an antibody against L. pneumophila has been generated from the naïve human HAL antibody libraries by phage-display for the first time. The panning was performed on whole bacterial cells in order to select antibodies that bind specifically to the cell surface of untreated Legionella. The bacterial cell wall component lipopolysaccharide (LPS) was identified as the target structure. Specific binding to the important pathogenic L. pneumophila strains Corby, Philadelphia-1 and Knoxville was observed, while no binding was detected to seven members of the families Enterobacteriaceae, Pseudomonadaceae or Clostridiaceae. Production of this antibody in the recombinant scFv-Fc format using either a murine or a human Fc part allowed the set-up of a sandwich-ELISA for detection of Legionella cells. The scFv-Fc construct proved to be very stable, even when stored for several weeks at elevated temperatures. A sensitivity limit of 4,000 cells was achieved. The scFv-Fc antibody pair was integrated on a biosensor, demonstrating the specific and fast detection of L. pneumophila on a portable device. With this system, 10,000 Legionella cells were detected within 35 min. Combined with a water filtration/concentration system, this antibody may be developed into a promising reagent for rapid on-site Legionella monitoring.