• Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.

      Volckmar, Julia; Gereke, Marcus; Ebensen, Thomas; Riese, Peggy; Philipsen, Lars; Lienenklaus, Stefan; Wohlleber, Dirk; Klopfleisch, Robert; Stegemann-Koniszewski, Sabine; Müller, Andreas J; et al. (2017-03-07)
      Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection.
    • Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes.

      Pogozhykh, Denys; Eicke, Dorothee; Gryshkov, Oleksandr; Wolkers, Willem F; Schulze, Kai; Guzmán, Carlos A; Blasczyk, Rainer; Figueiredo, Constança; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-16)
      Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.
    • TRANSFAC: a database on transcription factors and their DNA binding sites.

      Wingender, E; Dietze, P; Karas, H; Knüppel, R (1996-01-01)
    • TRANSVAC workshop on standardisation and harmonisation of analytical platforms for HIV, TB and malaria vaccines: 'how can big data help?'.

      Dutruel, Céline; Thole, Jelle; Geels, Mark; Mollenkopf, Hans-Joachim; Ottenhoff, Tom; Guzman, Carlos A; Fletcher, Helen A; Leroy, Odile; Kaufmann, Stefan H E; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2014-07-31)
      High-throughput analyses of RNA and protein expression are increasingly used for better understanding of vaccine-induced immunity and protection against infectious disease. With an increasing number of vaccine candidates in clinical development, it is timely to consider standardisation and harmonisation of sample collection, storage and analysis to ensure results of highest quality from these precious samples. These challenges were discussed by a group of international experts during a workshop organised by TRANSVAC, a European Commission-funded Research Infrastructure project. The main conclusions were: Platforms are rarely standardised for use in preclinical and clinical studies. Coordinated efforts should continue to harmonise the experimental set up of these studies, as well as the establishment of internal standards and controls. This will ensure comparability, efficiency and feasibility of the global analyses performed on preclinical and clinical data sets.
    • Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway.

      Lirussi, Darío; Ebensen, Thomas; Schulze, Kai; Trittel, Stephanie; Duran, Veronica; Liebich, Ines; Kalinke, Ulrich; Guzmán, Carlos Alberto; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-07-19)
      Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8(+) cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.
    • Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice.

      Retamal-Díaz, Angello; Riquelme-Neira, Roberto; Sáez, Darwin; Rivera, Alejandra; Fernández, Pablo; Cabrera, Alex; Guzmán, Carlos A; Oñate, Angel; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-11)
      This study was conducted to evaluate the immunogenicity and protective efficacy of a DNA vaccine encoding Brucella abortus Cu,Zn superoxide dismutase (SOD) using the Toll-like receptor 2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPPcysMPEG) as an adjuvant. Intranasal coadministration of BPPcysMPEG with a plasmid carrying the SOD-encoding gene (pcDNA-SOD) into BALB/c mice elicited antigen-specific humoral and cellular immune responses. Humoral responses were characterized by the stimulation of IgG2a and IgG1 and by the presence of SOD-specific secretory IgA in nasal and bronchoalveolar lavage fluids. Furthermore, T-cell proliferative responses and increased production of gamma interferon were also observed upon splenocyte restimulation with recombinant SOD. Cytotoxic responses were also stimulated, as demonstrated by the lysis of RB51-SOD-infected J774.A1 macrophages by cells recovered from immunized mice. The pcDNA-SOD/BPPcysMPEG formulation induced improved protection against challenge with the virulent strain B. abortus 2308 in BALB/c mice over that provided by pcDNA-SOD, suggesting the potential of this vaccination strategy against Brucella infection.
    • Vaccines against typhoid fever.

      Guzman, Carlos A; Borsutzky, Stefan; Griot-Wenk, Monika; Metcalfe, Ian C; Pearman, Jon; Collioud, Andre; Favre, Didier; Dietrich, Guido; Vaccine Research Group, Division of Microbiology, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, Braunschweig, Germany. (2006-05-01)
      Because of high infectivity and significant disease burden, typhoid fever constitutes a major global health problem. Implementation of adequate food handling practices and establishment of safe water supplies are the cornerstone for the development of an effective prevention program. However, vaccination against typhoid fever remains an essential tool for the effective management of this disease. Currently, there are two well tolerated and effective licensed vaccines. One is based on defined subunit virulence (Vi) polysaccharide antigen and can be administered either intramuscularly or subcutaneously and the other is based on the use of live attenuated bacteria for oral administration. The advantages and disadvantages of the various approaches taken in the development of a vaccine against typhoid fever are discussed, along with the potential for future vaccine candidates.
    • Vaccines: from empirical development to rational design.

      Rueckert, Christine; Guzmán, Carlos A; Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. (2012-11)
      Infectious diseases are responsible for an overwhelming number of deaths worldwide and their clinical management is often hampered by the emergence of multi-drug-resistant strains. Therefore, prevention through vaccination currently represents the best course of action to combat them. However, immune escape and evasion by pathogens often render vaccine development difficult. Furthermore, most currently available vaccines were empirically designed. In this review, we discuss why rational design of vaccines is not only desirable but also necessary. We introduce recent developments towards specifically tailored antigens, adjuvants, and delivery systems, and discuss the methodological gaps and lack of knowledge still hampering true rational vaccine design. Finally, we address the potential and limitations of different strategies and technologies for advancing vaccine development.
    • The Viable but Nonculturable State and Starvation Are Different Stress Responses of Enterococcus faecalis, as Determined by Proteome Analysis

      Heim, Sabina; Del Mar Lleo, Maria; Bonato, Barbara; Guzman, Carlos A.; Canepari, Pietro (American Society for Microbiology, 2002-12)
    • Virus Irradiation and COVID-19 Disease

      Durante, Marco; Schulze, Kai; Incerti, Sebastien; Francis, Ziad; Zein, Sara; Guzmán, Carlos Alberto; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-10-20)
      Virus irradiation has been performed for many decades for basic research studies, sterilization, and vaccine development. The COVID-19 outbreak is currently causing an enormous effort worldwide for finding a vaccine against coronavirus. High doses of γ-rays can be used for the development of vaccines that exploit inactivated virus. This technique has been gradually replaced by more practical methods, in particular the use of chemicals, but irradiation remains a simple and effective method used in some cases. The technique employed for inactivating a virus has an impact on its ability to induce an adaptive immune response able to confer effective protection. We propose here that accelerated heavy ions can be used to inactivate SARS-CoV-2 viruses with small damage to the spike proteins of the envelope and can then provide an intact virion for vaccine development.
    • Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the hepatitis B surface antigen.

      Lünsdorf, Heinrich; Gurramkonda, Chandrasekhar; Adnan, Ahmad; Khanna, Navin; Rinas, Ursula; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2011-06-26)
      A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.
    • Whole-Genome Enrichment Provides Deep Insights into Vibrio cholerae Metagenome from an African River.

      Vezzulli, L; Grande, C; Tassistro, G; Brettar, I; Höfle, M G; Pereira, R P A; Mushi, D; Pallavicini, A; Vassallo, P; Pruzzo, C; et al. (2016-11-25)
      The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next-generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (<50 genome unit/L) from natural water collected in the Morogoro river (Tanzania). The protocol is based on the use of biotinylated RNA baits for target enrichment of V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on an Illumina MiSeq platform. Up to 1.8 × 10(7) bp (4.5× mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices, this study provides a 'proof of concept' on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments.