• Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice.

      Skrnjug, Ivana; Guzmán, Carlos Alberto; Ruecker, Christine; Helmholtz Cente for infection research,Inhoffenstr. 7, D38124 Braunschweig, Germany. (2014)
      The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity - a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.
    • Deletion of Irf3 and Irf7 Genes in Mice Results in Altered Interferon Pathway Activation and Granulocyte-Dominated Inflammatory Responses to Influenza A Infection.

      Hatesuer, Bastian; Hoang, Hang Thi Thu; Riese, Peggy; Trittel, Stephanie; Gerhauser, Ingo; Elbahesh, Husni; Geffers, Robert; Wilk, Esther; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      The interferon (IFN) pathway plays an essential role in the innate immune response following viral infections and subsequent shaping of adaptive immunity. Infections with influenza A viruses (IAV) activate the IFN pathway after the recognition of pathogen-specific molecular patterns by respective pattern recognition receptors. The IFN regulatory factors IRF3 and IRF7 are key players in the regulation of type I and III IFN genes. In this study, we analyzed the role of IRF3 and IRF7 for the host response to IAV infections in Irf3-/-, Irf7-/-, and Irf3-/-Irf7-/- knockout mice. While the absence of IRF3 had only a moderate impact on IFN expression, deletion of IRF7 completely abolished IFNα production after infection. In contrast, lack of both IRF3 and IRF7 resulted in the absence of both IFNα and IFNβ after IAV infection. In addition, IAV infection of double knockout mice resulted in a strong increase of mortality associated with a massive influx of granulocytes in the lung and reduced activation of the adaptive immune response.
    • Development and characterization of attenuated metabolic mutants of Bordetella bronchiseptica for applications in vaccinology.

      Yevsa, Tetyana; Ebensen, Thomas; Fuchs, Barbara; Zygmunt, Beata; Libanova, Rimma; Gross, Roy; Schulze, Kai; Guzmán, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. (2013-01)
      Bordetella bronchiseptica is an important pathogen causing a number of veterinary respiratory syndromes in agriculturally important and food-producing confinement-reared animals, resulting in great economic losses annually amounting to billions of euros worldwide. Currently available live vaccines are incompletely satisfactory in terms of efficacy and safety. An efficient vaccine for livestock animals would allow reducing the application of antibiotics, thereby preventing the massive release of pharmaceuticals into the environment. Here, we describe two new potential vaccine strains based on the BB7865 strain. Two independent attenuating mutations were incorporated by homologous recombination in order to make negligible the risk of recombination and subsequent reversion to the virulent phenotype. The mutations are critical for bacterial metabolism, resistance to oxidative stress, intracellular survival and in vivo persistence. The resulting double mutants BB7865 risA aroA and BB7865 risA dapE were characterized as promising vaccine candidates, which are able to confer protection against colonization of the lower respiratory tract after sublethal challenge with the wild-type strain.
    • Dynamic changes in viral population structure and compartmentalization during chronic hepatitis C virus infection in children.

      Gismondi, María Inés; Díaz Carrasco, Juan María; Valva, Pamela; Becker, Pablo Daniel; Guzmán, Carlos Alberto; Campos, Rodolfo Héctor; Preciado, María Victoria; Helmholtz Centre for infection research, D-38124 Braunschweig, Germany (2013-12)
      Classic phylogenetic and modern population-based clustering methods were used to analyze hepatitis C virus (HCV) evolution in plasma and to assess viral compartmentalization within peripheral blood mononuclear cells (PBMCs) in 6 children during 3.2-9.6yr of follow-up. Population structure analysis of cloned amplicons encompassing hypervariable region 1 led to the distinction of two evolutionary patterns, one highly divergent and another one genetically homogeneous. Viral adaptability was reflected by co-evolution of viral communities switching rapidly from one to another in the context of divergence and stability associated with highly homogeneous communities which were replaced by new ones after long periods. Additionally, viral compartmentalization of HCV in PBMCs was statistically demonstrated, suggesting their role as a pool of genetic variability. Our results support the idea of a community-based structure of HCV viral populations during chronic infection and highlight a role of the PBMC compartment in the persistence of such structure.
    • Efficient nanoparticle-mediated needle-free transcutaneous vaccination via hair follicles requires adjuvantation.

      Mittal, Ankit; Schulze, Kai; Ebensen, Thomas; Weißmann, Sebastian; Hansen, Steffi; Lehr, Claus-Michael; Guzmán, Carlos A; Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. (2015-01)
      Trans-follicular (TF) vaccination has recently been studied as a unique route for non-invasive transcutaneous vaccination. The present study aims to extensively characterize the immune responses triggered by TF vaccination using ovalbumin loaded chitosan-PLGA (poly lactic-co-glycolic acid) nanoparticles without skin pre-treatment to preserve skin integrity. The impact of formulation composition i.e. antigenic solution or antigen-loaded nanoparticles with or without adjuvant [bis-(3',5')-cyclic dimeric adenosine monophosphate] on immune response quality following TF immunization was analyzed and compared with immune responses obtained after tape stripping the skin. The results presented in this study confirm the ability of nanoparticle based vaccine formulations to deliver antigen across the intact skin via the follicular route, but at the same time demonstrate the necessity to include adjuvants to generate efficient antigen-specific humoral and cellular immune responses.
    • Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV.

      Daenthanasanmak, Anusara; Salguero, Gustavo; Sundarasetty, Bala Sai; Waskow, Claudia; Cosgun, Kadriye Nehir; Guzman, Carlos A; Riese, Peggy; Gerasch, Laura; Schneider, Andreas; Ingendoh, Alexandra; et al. (2015)
      Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 ("SmyleDCpp65"). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4(+) and CD8(+) T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34(+) cells obtained from PB and CB were transplanted into irradiated NOD.Rag1(-/-).IL2γc(-/-) mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8(+) T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4(+)/CD8(+) T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.
    • Engineered trivalent immunogen adjuvanted with a STING agonist confers protection against Trypanosoma cruzi infection.

      Sanchez Alberti, Andrés; Bivona, Augusto E; Cerny, Natacha; Schulze, Kai; Weißmann, Sebastian; Ebensen, Thomas; Morales, Celina; Padilla, Angel M; Cazorla, Silvia I; Tarleton, Rick L; et al. (2017)
      The parasite Trypanosoma cruzi is the causative agent of Chagas disease, a potentially life-threatening infection that represents a major health problem in Latin America. Several characteristics of this protozoan contribute to the lack of an effective vaccine, among them: its silent invasion mechanism, T. cruzi antigen redundancy and immunodominance without protection. Taking into account these issues, we engineered Traspain, a chimeric antigen tailored to present a multivalent display of domains from key parasitic molecules, combined with stimulation of the STING pathway by c-di-AMP as a novel prophylactic strategy. This formulation proved to be effective for the priming of functional humoral responses and pathogen-specific CD8+ and CD4+ T cells, compatible with a Th1/Th17 bias. Interestingly, vaccine effectiveness assessed across the course of infection, showed a reduction in parasite load and chronic inflammation in different proof of concept assays. In conclusion, this approach represents a promising tool against parasitic chronic infections.
    • Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

      Fey, Axel; Eichler, Stefan; Flavier, Sébastien; Christen, Richard; Höfle, Manfred G.; Guzmán, Carlos A. (American Society for Microbiology, 2004-06)
    • Evaluation of the sublingual route for administration of influenza H5N1 virosomes in combination with the bacterial second messenger c-di-GMP.

      Pedersen, Gabriel Kristian; Ebensen, Thomas; Gjeraker, Ingrid Hjetland; Svindland, Signe; Bredholt, Geir; Guzmán, Carlos Alberto; Cox, Rebecca Jane; The Gade Institute, University of Bergen, Norway. gabriel.pedersen@gades.uib.no (2011)
      Avian influenza A H5N1 is a virus with pandemic potential. Mucosal vaccines are attractive as they have the potential to block viruses at the site of entry, thereby preventing both disease and further transmission. The intranasal route is safe for the administration of seasonal live-attenuated influenza vaccines, but may be less suitable for administration of pandemic vaccines. Research into novel mucosal routes is therefore needed. In this study, a murine model was used to compare sublingual administration with intranasal and intramuscular administration of influenza H5N1 virosomes (2 µg haemagglutinin; HA) in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP). We found that sublingual immunisation effectively induced local and systemic H5N1-specific humoral and cellular immune responses but that the magnitude of response was lower than after intranasal administration. However, both the mucosal routes were superior to intramuscular immunisation for induction of local humoral and systemic cellular immune responses including high frequencies of splenic H5N1-specific multifunctional (IL-2+TNF-α+) CD4+ T cells. The c-di-GMP adjuvanted vaccine elicited systemic haemagglutination inhibition (HI) antibody responses (geometric mean titres ≥ 40) both when administered sublingually, intranasally and inramuscularly. In addition, salivary HI antibodies were elicited by mucosal, but not intramuscular vaccination. We conclude that the sublingual route is an attractive alternative for administration of pandemic influenza vaccines.
    • Evolution of hepatitis C virus hypervariable region 1 in immunocompetent children born to HCV-infected mothers.

      Gismondi, M I; Becker, P D; Díaz Carrasco, J M; Guzmán, C A; Campos, R H; Preciado, M V; Laboratorio de Biología Molecular, División Patología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina. (2009-05)
      Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d(N)/d(S) rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S))] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.
    • Exploitation of prokaryotic expression systems based on the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2 for biotechnological applications.

      Becker, Pablo D; Royo, Jose L; Guzman, Carlos A; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-15)
      Expression vectors appear to be an indispensable tool for both biological studies and biotechnological applications. Controlling gene overexpression becomes a critical issue when protein production is desired. In addition to several aspects regarding toxicity or plasmid instability, tight control of gene expression is an essential factor in biotechnological processes. Thus, the search for better-controlled circuits is an important issue among biotechnologists. Traditionally, expression systems involve a single regulatory protein operating over a target promoter. However, these circuits are limited on their induction ratios (e.g., by their restriction in the maximal expression capacity, by their leakiness under non-induced conditions). Due to these limitations, regulatory cascades, which are far more efficient, are necessary for biotechnological applications. Thus, regulatory circuits with two modules operating in cascade offer a significant advantage. In this review, we describe the regulatory cascade based on two salicylate-responsive transcriptional regulators of Pseudomonas putida (nahR/P(sal)::xylS2), its properties, and contribution to a tighter control over heterologous gene expression in different applications.Nowadays, heterologous expression has been proven to be an indispensable tool for tackling basic biological questions, as well as for developing biotechnological applications. As the nature of the protein of interest becomes more complex, biotechnologists find that a tight control of gene expression is a key factor which conditions the success of the downstream purification process, as well as the interpretation of the results in other type of studies. Fortunately, different expression systems can be found in the market, each of them with their own pros and cons. In this review we discuss the exploitation of prokaryotic expression systems based on a promising expression system, the salicylate-dependent control circuit encompassing nahR/P(sal)::xylS2, as well as some of the improvements that have been done on this system to exploit it more efficiently in the context of both biotechnological applications and basic research.
    • Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

      Becker, Pablo D; Nörder, Miriam; Weissmann, Sebastian; Ljapoci, Ronny; Erfle, Volker; Drexler, Ingo; Guzmán, Carlos Alberto; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-07-22)
      Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.
    • Gene expression profiles of T cells from hepatitis E virus infected patients in acute and resolving phase.

      TrehanPati, Nirupma; Sukriti, Sukriti; Geffers, Robert; Hissar, Syed; Riese, Peggy; Toepfer, Tanja; Guzman, Carlos A; Sarin, Shiv Kumar; Institute of Liver and Biliary Sciences, D-1, Vasant Kunj, 110 070, New Delhi, India. trehanpati@gmail.com (2011-06)
      Approximately 50% of acute viral hepatitis in young adults and in pregnant women is due to hepatitis E virus (HEV) infection in developing countries. T cell-mediated immune injury probably plays a key role in the pathogenesis of acute hepatitis illness. However, there is a paucity of data on the global gene expression programs activated on T cells, which are subsequently responsible for T cell recruitment to the liver and triggering of immune injury.
    • Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

      Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; et al. (2015)
      Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.
    • Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice.

      Becker, Pablo D; Legrand, Nicolas; van Geelen, Caroline M M; Noerder, Miriam; Huntington, Nicholas D; Lim, Annick; Yasuda, Etsuko; Diehl, Sean A; Scheeren, Ferenc A; Ott, Michael; et al. (2010)
      Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique.
    • Hepatitis C Virus Isolates from Argentina Disclose a Novel Genotype 1-Associated Restriction Pattern

      Gismondi, María Inés; Staendner, Lothar Heinrich; Grinstein, Saúl; Guzmán, Carlos Alberto; Preciado, María Victoria (American Society for Microbiology, 2004-03)
    • High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA.

      Kahlisch, Leila; Henne, Karsten; Draheim, Josefin; Brettar, Ingrid; Höfle, Manfred G; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany. (2010-09)
      Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.
    • Identification of a Thiomicrospira denitrificans-Like Epsilonproteobacterium as a Catalyst for Autotrophic Denitrification in the Central Baltic Sea†

      Brettar, Ingrid; Labrenz, Matthias; Flavier, Sébastien; Bötel, Julia; Kuosa, Harri; Christen, Richard; Höfle, Manfred G. (American Society for Microbiology, 2006-02)