Recent Submissions

  • PD-1 expression affects cytokine production by ILC2 and is influenced by peroxisome proliferator-activated receptor-γ.

    Batyrova, Banu; Luwaert, Fien; Maravelia, Panagiota; Miyabayashi, Yuria; Vashist, Neha; Stark, Julian M; Soori, Sara Y; Tibbitt, Christopher A; Riese, Peggy; Coquet, Jonathan M; et al. (Wiley, 2019-11-19)
    Innate lymphoid cells (ILCs) can provide early cytokine help against a variety of pathogens in the lungs and gastrointestinal tract. Type 2 ILC (ILC2) are comparable to T helper 2 cells found in the adaptive immune system, which secrete cytokines such as interleukin 5 (IL-5) and IL-13 and have been found to play roles in host defense against helminth infections and in allergic responses. Recent studies have identified that programmed cell death protein 1 (PD-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are highly expressed by ILC2. We examined whether PD-1 plays a role in ILC2 function and whether there was any connection between PD-1 and PPAR-γ METHODS: To ensure that only innate immune cells were present, ILC2 cells were examined from RAG1-/- and PD-1-/- xRAG1-/- mice under steady-state or following inoculation with IL-33. We also tested ILC2 generated from bone marrow of RAG1-/- and PD-1-/- xRAG1-/- mice for their production of cytokines. These in vitro-derived ILC2 were also exposed to agonist and antagonist of PPAR-γ.
  • Key features and homing properties of NK cells in the liver are shaped by activated iNKT cells.

    Trittel, Stephanie; Chambers, Benedict J; Heise, Ulrike; Guzmán, Carlos A; Riese, Peggy; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-11-08)
    The contribution of natural killer (NK) cells to the clearance of hepatic viral infections is well recognized. The recently discovered heterogeneity of NK cell populations renders them interesting targets for immune interventions. Invariant natural killer T (iNKT) cells represent a key interaction partner for hepatic NK cells. The present study addressed whether characteristics of NK cells in the liver can be shaped by targeting iNKT cells. For this, the CD1d-binding pegylated glycolipid αGalCerMPEG was assessed for its ability to modulate the features of NK cells permanently or transiently residing in the liver. In vivo administration resulted in enhanced functionality of educated and highly differentiated CD27+ Mac-1+ NK cells accompanied by an increased proliferation. Improved liver homing was supported by serum-derived and cellular factors. Reduced viral loads in a mCMV infection model confirmed the beneficial effect of NK cells located in the liver upon stimulation with αGalCerMPEG. Thus, targeting iNKT cell-mediated NK cell activation in the liver represents a promising approach for the establishment of liver-directed immune interventions.
  • Genomic Variations Underlying Speciation and Niche Specialization of Shewanella baltica.

    Deng, Jie; Auchtung, Jennifer M; Konstantinidis, Konstantinos T; Brettar, Ingrid; Höfle, Manfred G; Tiedje, James M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Society of Microbiology, 2019-10-15)
    Shewanella baltica was the dominant culturable nitrate-reducing bacterium in the eutrophic and strongly stratified Baltic Sea in the 1980s, where it primarily inhabited the oxic-anoxic transition zone. The genomic structures of 46 of these isolates were investigated through comparative genomic hybridization (CGH), which revealed a gradient of genomic similarity, ranging from 65% to as high as 99%. The core genome of the S. baltica species was enriched in anaerobic respiration-associated genes. Auxiliary genes, most of which locate within a few genomic islands (GIs), were nonuniformly distributed among the isolates. Specifically, hypothetical and mobile genetic element (MGE)-associated genes dominated intraclade gene content differences, whereas gain/loss of functional genes drove gene content differences among less related strains. Among the major S. baltica clades, gene signatures related to specific redox-driven and spatial niches within the water column were identified. For instance, genes involved in anaerobic respiration of sulfur compounds may provide key adaptive advantages for clade A strains in anoxic waters where sulfur-containing electron acceptors are present. Genes involved in cell motility, in particular, a secondary flagellar biosynthesis system, may be associated with the free-living lifestyle by clade E strains. Collectively, this study revealed characteristics of genome variations present in the water column and active speciation of S. baltica strains, driven by niche partitioning and horizontal gene transfer (HGT).IMPORTANCE Speciation in nature is a fundamental process driving the formation of the vast microbial diversity on Earth. In the central Baltic Sea, the long-term stratification of water led to formation of a large-scale vertical redoxcline that provided a gradient of environmental niches with respect to the availability of electron acceptors and donors. The region was home to Shewanella baltica populations, which composed the dominant culturable nitrate-reducing bacteria, particularly in the oxic-anoxic transition zone. Using the collection of S. baltica isolates as a model system, genomic variations showed contrasting gene-sharing patterns within versus among S. baltica clades and revealed genomic signatures of S. baltica clades related to redox niche specialization as well as particle association. This study provides important insights into genomic mechanisms underlying bacterial speciation within this unique natural redoxcline.
  • Vaccine Vectors Harnessing the Power of Cytomegaloviruses.

    Ynga-Durand, Mario Alberto; Dekhtiarenko, Iryna; Cicin-Sain, Luka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-10-17)
    Cytomegalovirus (CMV) species have been gaining attention as experimental vaccine vectors inducing cellular immune responses of unparalleled strength and protection. This review outline the strengths and the restrictions of CMV-based vectors, in light of the known aspects of CMV infection, pathogenicity and immunity. We discuss aspects to be considered when optimizing CMV based vaccines, including the innate immune response, the adaptive humoral immunity and the T-cell responses. We also discuss the antigenic epitopes presented by unconventional major histocompatibility complex (MHC) molecules in some CMV delivery systems and considerations about routes for delivery for the induction of systemic or mucosal immune responses. With the first clinical trials initiating, CMV-based vaccine vectors are entering a mature phase of development. This impetus needs to be maintained by scientific advances that feed the progress of this technological platform.
  • Mucosal CD8+ T cell responses induced by an MCMV based vaccine vector confer protection against influenza challenge.

    Zheng, Xiaoyan; Oduro, Jennifer D; Boehme, Julia D; Borkner, Lisa; Ebensen, Thomas; Heise, Ulrike; Gereke, Marcus; Pils, Marina C; Krmpotic, Astrid; Guzmán, Carlos A; et al. (PLOS, 2019-09-01)
    Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.
  • Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine.

    Perche, Federico; Clemençon, Rudy; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Pichon, Chantal; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-09-06)
    Nucleic acid vaccination relies on injecting DNA or RNA coding antigen(s) to induce a protective immune response. RNA vaccination is being increasingly used in preclinical and clinical studies. However, few delivery systems have been reported for in vivo delivery of RNA of different sizes. Using a tripartite formulation with RNA, cationic polymer, and anionic liposomes, we were able to encapsulate RNA into neutral lipopolyplexes (LPPs). LPPs were stable in vitro and successfully delivered conventional RNA and replicative RNA to dendritic cells in cellulo. Their injection led to reporter gene expression in mice. Finally, administration of LPP-Replicon RNA (RepRNA) led to an adaptive immune response against the antigen coded by the RepRNA. Accordingly, LPPs may represent a universal formulation for RNA delivery.
  • In vivo Neutralization of Pro-inflammatory Cytokines During Secondary Streptococcus pneumoniae Infection Post Influenza A Virus Infection

    Sharma-Chawla, Niharika; Stegemann-Koniszewski, Sabine; Christen, Henrike; Boehme, Julia D.; Kershaw, Olivia; Schreiber, Jens; Guzmán, Carlos A.; Bruder, Dunja; Hernandez-Vargas, Esteban A.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers Media SA, 2019-08-14)
    An overt pro-inflammatory immune response is a key factor contributing to lethal pneumococcal infection in an influenza pre-infected host and represents a potential target for therapeutic intervention. However, there is a paucity of knowledge about the level of contribution of individual cytokines. Based on the predictions of our previous mathematical modeling approach, the potential benefit of IFN-γ- and/or IL-6-specific antibody-mediated cytokine neutralization was explored in C57BL/6 mice infected with the influenza A/PR/8/34 strain, which were subsequently infected with the Streptococcus pneumoniae strain TIGR4 on day 7 post influenza. While single IL-6 neutralization had no effect on respiratory bacterial clearance, single IFN-γ neutralization enhanced local bacterial clearance in the lungs. Concomitant neutralization of IFN-γ and IL-6 significantly reduced the degree of pneumonia as well as bacteremia compared to the control group, indicating a positive effect for the host during secondary bacterial infection. The results of our model-driven experimental study reveal that the predicted therapeutic value of IFN-γ and IL-6 neutralization in secondary pneumococcal infection following influenza infection is tightly dependent on the experimental protocol while at the same time paving the way toward the development of effective immune therapies.
  • Invariant NKT Cell-Mediated Modulation of ILC1s as a Tool for Mucosal Immune Intervention.

    Trittel, Stephanie; Vashist, Neha; Ebensen, Thomas; Chambers, Benedict J; Guzmán, Carlos A; Riese, Peggy; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
    Non-NK group 1 innate lymphoid cells (ILC1s), mainly investigated in the mucosal areas of the intestine, are well-known to contribute to anti-parasitic and anti-bacterial immune responses. Recently, our group revealed that lung ILC1s become activated during murine influenza infection, thereby contributing to viral clearance. In this context, worldwide seasonal influenza infections often result in severe disease outbreaks leading to high morbidity and mortality. Therefore, new immune interventions are urgently needed. In contrast to NK cells, the potential of non-NK ILC1s to become functionally tailored by immune modulators to contribute to the combat against mucosal-transmitted viral pathogens has not yet been addressed. The present study aimed at assessing the potential of ILC1s to become modulated by iNKT cells activated through the CD1d agonist αGalCerMPEG. Our results demonstrate an improved functional responsiveness of murine lung and splenic ILC1s following iNKT cell stimulation by the mucosal route, as demonstrated by enhanced surface expression of TNF-related apoptosis-inducing ligand (TRAIL), CD49a and CD28, and increased secretion of IFNγ. Interestingly, iNKT cell stimulation also induced the expression of the immune checkpoint molecules GITR and CTLA-4, which represent crucial points of action for immune regulation. An in vivo influenza infection model revealed that intranasal activation of ILC1s by αGalCerMPEG contributed to increased viral clearance as shown by reduced viral loads in the lungs. The findings that ILC1s can become modulated by mucosally activated iNKT cells in a beneficial manner emphasize their up to now underestimated potential and renders them to be considered as targets for novel immune interventions.
  • The STING activator c-di-AMP exerts superior adjuvant properties than the formulation poly(I:C)/CpG after subcutaneous vaccination with soluble protein antigen or DEC-205-mediated antigen targeting to dendritic cells.

    Volckmar, Julia; Knop, Laura; Stegemann-Koniszewski, Sabine; Schulze, Kai; Ebensen, Thomas; Guzmán, Carlos A; Bruder, Dunja; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2019-08-14)
    Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8+ memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches.
  • Establishment of an induced memory response in Pseudomonas aeruginosa during infection of a eukaryotic host.

    Kordes, Adrian; Grahl, Nora; Koska, Michal; Preusse, Matthias; Arce-Rodriguez, Alejandro; Abraham, Wolf-Rainer; Kaever, Volkhard; Häussler, Susanne; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer-Nature, 2019-08-01)
    In a given habitat, bacterial cells often experience recurrent exposures to the same environmental stimulus. The ability to memorize the past event and to adjust current behaviors can lead to efficient adaptation to the recurring stimulus. Here we demonstrate that the versatile bacterium Pseudomonas aeruginosa adopts a virulence phenotype after serial passage in the invertebrate model host Galleria mellonella. The virulence phenotype was not linked to the acquisition of genetic variations and was sustained for several generations, despite cultivation of the ex vivo virulence-adapted P. aeruginosa cells under rich medium conditions in vitro. Transcriptional reprogramming seemed to be induced by a host-specific food source, as reprogramming was also observed upon cultivation of P. aeruginosa in rich medium supplemented with polyunsaturated long-chain fatty acids. The establishment of induced memory responses adds a time dimension and seems to fill the gap between long-term evolutionary genotypic adaptation and short-term induced individual responses. Efforts to unravel the fundamental mechanisms that underlie the carry-over effect to induce such memory responses will continue to be of importance as hysteretic behavior can serve survival of bacterial populations in changing and challenging habitats.
  • Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

    Contreras, Nico A; Sitnik, Katarzyna M; Jeftic, Ilija; Coplen, Christopher Patrick; Čičin-Šain, Luka; Nikolich-Žugich, Janko; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-06-01)
    Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world's population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.
  • Systems Immunology Characterization of Novel Vaccine Formulations for Mycoplasma hyopneumoniae Bacterins.

    Matthijs, Anneleen M F; Auray, Gaël; Jakob, Virginie; García-Nicolás, Obdulio; Braun, Roman O; Keller, Irene; Bruggman, Rémy; Devriendt, Bert; Boyen, Filip; Guzman, Carlos A; et al. (Frontiers, 2019-01-01)
    We characterized five different vaccine candidates and a commercial vaccine in terms of safety, immunogenicity and using a systems vaccinology approach, with the aim to select novel vaccine candidates against Mycoplasma hyopneumoniae. Seven groups of six M. hyopneumoniae-free piglets were primo- and booster vaccinated with the different experimental bacterin formulations, the commercial vaccine Hyogen® as a positive control or PBS as a negative control. The experimental bacterin was formulated with cationic liposomes + c-di-AMP (Lipo_AMP), cationic liposomes + Toll-like receptor (TLR) 2/1, TLR7, and TLR9 ligands (TLR ligands; Lipo_TLR), micro-particles + TLR ligands (PLGA_TLR), squalene-in-water emulsion + TLR ligands (SWE_TLR), or DDA:TDB liposomes (Lipo_DDA:TDB). Lipo_DDA:TDB and Lipo_AMP were the most potent in terms of serum antibody induction, and Lipo_DDA:TDB, Lipo_AMP, and SWE_TLR significantly induced Th1 cytokine-secreting T-cells. Only PLGA_TLR appeared to induce Th17 cells, but was unable to induce serum antibodies. The transcriptomic analyses demonstrated that the induction of inflammatory and myeloid cell blood transcriptional modules (BTM) in the first 24 h after vaccination correlated well with serum antibodies, while negative correlations with the same modules were found 7 days post-vaccination. Furthermore, many cell cycle and T-cell BTM upregulated at day seven correlated positively with adaptive immune responses. When comparing the delivery of the identical TLR ligands with the three formulations, we found SWE_TLR to be more potent in the induction of an early innate immune response, while the liposomal formulation more strongly promoted late cell cycle and T-cell BTM. For the PLGA formulation we found signs of a delayed and weak perturbation of these BTM. Lipo_AMP was found to be the most potent vaccine at inducing a BTM profile similar to that correlating with adaptive immune response in this and other studies. Taken together, we identified four promising vaccine candidates able to induce M. hyopneumoniae-specific antibody and T-cell responses. In addition, we have adapted a systems vaccinology approach developed for human to pigs and demonstrated its capacity in identifying early immune signatures in the blood relating to adaptive immune responses. This approach represents an important step in a more rational design of efficacious vaccines for pigs.
  • Early primed KLRG1- CMV-specific T cells determine the size of the inflationary T cell pool.

    Baumann, Nicolas S; Welten, Suzanne P M; Torti, Nicole; Pallmer, Katharina; Borsa, Mariana; Barnstorf, Isabel; Oduro, Jennifer D; Cicin-Sain, Luka; Oxenius, Annette; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-05-01)
    Memory T cell inflation is a process in which a subset of cytomegalovirus (CMV) specific CD8 T cells continuously expands mainly during latent infection and establishes a large and stable population of effector memory cells in peripheral tissues. Here we set out to identify in vivo parameters that promote and limit CD8 T cell inflation in the context of MCMV infection. We found that the inflationary T cell pool comprised mainly high avidity CD8 T cells, outcompeting lower avidity CD8 T cells. Furthermore, the size of the inflationary T cell pool was not restricted by the availability of specific tissue niches, but it was directly related to the number of virus-specific CD8 T cells that were activated during priming. In particular, the amount of early-primed KLRG1- cells and the number of inflationary cells with a central memory phenotype were a critical determinant for the overall magnitude of the inflationary T cell pool. Inflationary memory CD8 T cells provided protection from a Vaccinia virus challenge and this protection directly correlated with the size of the inflationary memory T cell pool in peripheral tissues. These results highlight the remarkable protective potential of inflationary CD8 T cells that can be harnessed for CMV-based T cell vaccine approaches.
  • Circulating levels of the anti-oxidant indoleproprionic acid are associated with higher gut microbiome diversity.

    Menni, Cristina; Hernandez, Marisa Matey; Vital, Marius; Mohney, Robert P; Spector, Tim D; Valdes, Ana M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2019-04-29)
    The gut microbiome has recently emerged as an important regulator of insulin resistance and abdominal obesity. The tryptophan metabolite generated by the gut microbiome, indoleproprionic acid (IPA) has been shown to predict the onset of type 2 diabetes. IPA is a metabolite produced by gut microbes from dietary tryptophan that exhibits a high degree of inter-individual variation. The microbiome composition parameters that are associated with circulating levels of this potent anti-oxidant have however not been investigated to date in human populations. In 1018 middle-aged women from the TwinsUK cohort, we assessed the relationship between serum IPA levels and gut microbiome composition targeting the 16S rRNA gene. Microbiome alpha-diversity was positively correlated with serum indoleproprionic acid levels (Shannon Diversity: Beta[95%CI] = 0.19[0.13;0.25], P = 6.41 × 10-10) after adjustment for covariates. Sixteen taxa and 12 operational taxonomic units (OTUs) associated with IPA serum levels. Among these are positive correlations with the butyrate-producing Faecalibacterium prausnitzii, the class Mollicutes and the order RF39 of the Tenericutes, and Coprococcus Negative correlations instead were observed with Eubacterium dolichum previously shown to correlate with visceral fat mass and several genera in the Lachnospiraceae family such as Blautia and Ruminococcus previously shown to correlate with obesity. Microbiome composition parameters explained ~20% of the variation in circulating levels of IPA, whereas nutritional and host genetic parameters explained only ~4%. Our data confirm an association between IPA circulating levels and metabolic syndrome parameters and indicate that gut microbiome composition influences IPA levels.
  • Model-based analysis of influenza A virus replication in genetically engineered cell lines elucidates the impact of host cell factors on key kinetic parameters of virus growth.

    Laske, Tanja; Bachmann, Mandy; Dostert, Melanie; Karlas, Alexander; Wirth, Dagmar; Frensing, Timo; Meyer, Thomas F; Hauser, Hansjörg; Reichl, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PLOS, 2019-01-01)
    The best measure to limit spread of contagious diseases caused by influenza A viruses (IAVs) is annual vaccination. The growing global demand for low-cost vaccines requires the establishment of high-yield production processes. One possible option to address this challenge is the engineering of novel vaccine producer cell lines by manipulating gene expression of host cell factors relevant for virus replication. To support detailed characterization of engineered cell lines, we fitted an ordinary differential equation (ODE)-based model of intracellular IAV replication previously established by our group to experimental data obtained from infection studies in human A549 cells. Model predictions indicate that steps of viral RNA synthesis, their regulation and particle assembly and virus budding are promising targets for cell line engineering. The importance of these steps was confirmed in four of five single gene overexpression cell lines (SGOs) that showed small, but reproducible changes in early dynamics of RNA synthesis and virus release. Model-based analysis suggests, however, that overexpression of the selected host cell factors negatively influences specific RNA synthesis rates. Still, virus yield was rescued by an increase in the virus release rate. Based on parameter estimations obtained for SGOs, we predicted that there is a potential benefit associated with overexpressing multiple host cell genes in one cell line, which was validated experimentally. Overall, this model-based study on IAV replication in engineered cell lines provides a step forward in the dynamic and quantitative characterization of IAV-host cell interactions. Furthermore, it suggests targets for gene editing and indicates that overexpression of multiple host cell factors may be beneficial for the design of novel producer cell lines.
  • Antimicrobial agent susceptibilities of Legionella pneumophila MLVA-8 genotypes.

    Sharaby, Yehonatan; Nitzan, Orna; Brettar, Ingrid; Höfle, Manfred G; Peretz, Avi; Halpern, Malka; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature publishing group, 2019-04-16)
    Legionella pneumophila causes human lung infections resulting in severe pneumonia. High-resolution genotyping of L. pneumophila isolates can be achieved by multiple-locus variable-number tandem-repeat analysis (MLVA-8). Legionella infections in humans occur as a result of inhalation of bacteria-containing aerosols, thus, our aim was to study the antimicrobial susceptibilities of different MLVA-8 genotypes to ten commonly used antimicrobial agents in legionellosis therapy. Epidemiological cut-off values were determined for all antibiotics. Significant differences were found between the antimicrobial agents’ susceptibilities of the three studied environmental genotypes (Gt4, Gt6, and Gt15). Each genotype exhibited a significantly different susceptibility profile, with Gt4 strains (Sequence Type 1) significantly more resistant towards most studied antimicrobial agents. In contrast, Gt6 strains (also Sequence Type 1) were more susceptible to six of the ten studied antimicrobial agents compared to the other genotypes. Our findings show that environmental strains isolated from adjacent points of the same water system, exhibit distinct antimicrobial resistance profiles. These differences highlight the importance of susceptibility testing of Legionella strains. In Israel, the most extensively used macrolide for pneumonia is azithromycin. Our results point at the fact that clarithromycin (another macrolide) and trimethoprim with sulfamethoxazole (SXT) were the most effective antimicrobial agents towards L. pneumophila strains. Moreover, legionellosis can be caused by multiple L. pneumophila genotypes, thus, the treatment approach should be the use of combined antibiotic therapy. Further studies are needed to evaluate specific antimicrobial combinations for legionellosis therapy.
  • ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.

    Büssow, Konrad; Themann, Philipp; Luu, Sabine; Pentrowski, Paul; Harting, Claudia; Majewski, Mira; Vollmer, Veith; Köster, Mario; Grashoff, Martina; Zawatzky, Rainer; et al. (Plos, 2019-01-01)
    Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
  • The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.

    Ebensen, Thomas; Delandre, Simon; Prochnow, Blair; Guzmán, Carlos A; Schulze, Kai (2019-01-01)
  • Demarcated thresholds of tumor-specific CD8 T cells elicited by MCMV-based vaccine vectors provide robust correlates of protection.

    Beyranvand Nejad, Elham; Ratts, Robert B; Panagioti, Eleni; Meyer, Christine; Oduro, Jennifer D; Cicin-Sain, Luka; Früh, Klaus; van der Burg, Sjoerd H; Arens, Ramon; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2019-01-31)
    The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8 Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8 This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients.
  • An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

    Dubich, Tatyana; Lieske, Anna; Santag, Susann; Beauclair, Guillaume; Rückert, Jessica; Herrmann, Jennifer; Gorges, Jan; Büsche, Guntram; Kazmaier, Uli; Hauser, Hansjörg; et al. (2019-01-04)
    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo.

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