A Training Network for the Design of Synthetic Carbohydrate-Based Vaccines in the Fight against Multi-Drug Resistant Nosocomial Pathogen Acinetobacter baumannii (ACINETWORK)
The bacterium Acinetobacter baumannii (Ab) strains ATCC 17978 and ATCC 7961 has been used by several laboratories in mouse models of respiratory infection. The availability of pure and well-defined polysaccharide synthetic fragments of both species would be of great interest to study their role in the pathogenicity of the bacterium. Bacterial capsular polysaccharides found in strains ATCC 17961 and ATCC 17978 share the same pentasaccharide repeating unit, the only structural difference being a single O-acetylation occurring in ATCC 17978 and missing in ATCC 17961. The recruited DC will be primarily engaged in the 1) synthesis of the pentasaccharide repeating units of the polysaccharides of both ATCC 17978 and ATCC 17961 Ab strains, and 2) synthesis of di- and trisaccharide fragments subunits of both repeating units. The synthetic glycans will be chemically conjugated to different carriers (immunogenic proteins and nanoparticles) for immunoevaluation.
Synthesis of a panel of glycans from ATCC 17978 and ATCC 17961 Acinetobacter baumannii capsular polysaccharides.
1 • Dr. Kim Le Mai Hoang, Potsdam (GlycoUniverse, D) to be trained in automated synthesis of oligosaccharides;
2 • Dr. Sergio E. Moya, San Sebastian (CICbiomaGUNE, E) to be trained in synthesis and characterization of glycosylated supramolecular nanoparticles.
The recruited DC will be placed at the Department of Chemistry (https://eng.chimica.unimi.it/ecm/home), University of Milan, in the laboratory of Prof. Luigi Lay and under his supervision, with the co-supervision of Dr. Giuseppe D’Orazio. The recruited DC will be enrolled in the PhD programme in “Chemistry” which offers a broad range of courses on core chemistry disciplines and complementary skills. The laboratory of Prof. Lay is fully equipped with cutting-edge instrumentation for organic synthesis, including MW reactor, flow-chemistry devices, analytical and preparative chromatographic equipment. The host laboratory has state-of-the-art Internet connection with full access to the main literature databases (Reaxys, Sci-Finder, Web of Science) and on-line access to the primary literature sources. In addition, the host laboratory has full access to high field NMR facilities, HR-MS and MALDI spectrometers, FT-IR, UV, TEM, etc.
LAC-4 belongs to a group of 20 multidrug resistant clinical isolates of Acinetobacter baumannii (Ab) obtained from nosocomial outbreaks in Los Angeles County hospitals, identified as hypervirulent strain in a mouse model of intranasal infection in comparison to other clinical isolates and laboratory strains of Ab. Notably, the LAC-4 strain exhibits high serum resistance and reliably reproduces the most relevant features of human pulmonary Ab infection. The surface polysaccharide of LAC-4 strain is made up of a trisaccharide repeating unit which contains synthetically challenging monosaccharide residues, including 8epi-Legionaminic acid (the C-8 epimer of 5,7-di-N-acetyl-legionaminic acid). The latter belongs to the group of nonulosonic acids (also comprising sialic acid), a class of sugars considered to be a virulence factor within a wide variety of pathogenic bacteria. The major tasks of the recruited DC will be 1) the design and synthesis of 8epiLegionaminic acid building block, and 2) its incorporation into the full trisaccharide repeating unit of LAC-4 polysaccharide. The synthetic glycans will be chemically conjugated to different carriers (immunogenic proteins and nanoparticles) for immunoevaluation.
Synthesis of 8epi-Legionaminic acid and its incorporation into the trisaccharide repeating unit of LAC-4 Acinetobacter baumannii polysaccharide and larger structures.
1 • Prof. Barbara Richichi and Prof. Marco Marradi, Florence (University of Florence, I) to be trained in the conjugation of oligosaccharides to protein and gold nanoparticles;
2 • Dr. Kim Le Mai Hoang, Potsdam (GlycoUniverse, D) to be trained in automated synthesis of oligosaccharides.
The laboratory of Prof. Lay is fully equipped with cutting-edge instrumentation for organic synthesis, including MW reactor, flow-chemistry devices, analytical and preparative chromatographic equipment. The host laboratory has state-of-the-art Internet connection with full access to the main literature databases (Reaxys, Sci-Finder, Web of Science) and on-line access to the primary literature sources. In addition, the host laboratory has full access to high field NMR facilities, HR-MS and MALDI spectrometers, FT-IR, UV, TEM, etc.
The chemical conjugation of pathogen-associated saccharide antigens to immunogenic protein carriers is a prerequisite to rendering sugars capable of evoking persistent immunological memory and durable host protection. Gold nanoparticles (AuNPs) represent alternative multivalent carriers enabling the conjugation of different saccharide fragments targeting the same Acinetobacter baumannii (Ab) strain, or a combination of oligosaccharide antigens related to distinct bacterial strains. In the latter case the nanosystem shall include a T-helper peptide to ensure a B cell-mediated immunological memory. In this context, the main tasks of the recruited fellow will be 1) site-selective conjugation of Ab synthetic oligosaccharides available from the consortium to the immunogenic protein carrier cross reacting material 197 (CRM197), and 2) synthesis of glycosylated AuNPs bearing Ab synthetic oligosaccharides. All glycoconjugates obtained will be used to aid the identification of the minimum binding epitope recognised by anti-Ab antibodies.
1) Synthesis of protein conjugates of Acinetobacter baumannii saccharide fragments with different carbohydrate:protein ratio.
2) Surface functionalization of gold nanoparticles with Acinetobacter baumannii synthetic oligosaccharides.
1 • Dr. Laura Polito, Milan (National Research Council, SCITEC Institute, I) to be trained in microfluidic production of gold nanoparticles and their functionalization with synthetic oligosaccharides;
2 • Dr. Antonio J. Martín Galiano, Madrid (Instituto de Salud CARLOS III, E) to be trained in ELISA and in vivo immunity test.
The host laboratories (DICUS–UNIFI) are equipped with modern equipment for organic synthesis and nanomaterials, including 400MHz NMR spectroscopy facilities, a state of the art cryo-electron microscope (cryo-EM), polarimeter, DLS, microwave reactors, HPLCs, FT-IR, and mass (GC/MS, ESI/MS) spectrometers, UV-Vis spectrophotometers, freeze dryers, and centrifuges. Full access to the literature databases (Reaxys, Web of Science, ScFinder) and on-line access to the primary lit.
The bacterium Acinetobacter baumannii (Ab) strain ATCC 19606 has a lipopolysaccharide (LPS) core region composed by a hexasaccharide structure which has been proposed to be highly specific to virulent Ab species. Notably, the core region of SMAL Ab strain is identical to the major LPS component of strain ATCC 19606. These structures hence represent promising candidates as carbohydrate antigens for glycoconjugate vaccine development against Ab. In this project, a series of glycan epitopes belonging to the LPS core region of ATCC 19606 will be prepared using solution phase synthesis and automated glycan assembly. The synthetic glycans will be used to screen human sera for anti-glycan antibodies in order to define a vaccine lead. Neoglycoconjugates will be prepared and tested in vivo. The recruited DC will be primarily engaged in the 1) synthesis of oligosaccharide fragments of Ab ATCC 19606 LPS core region, including the hexasaccharide repeating unit, and 2) chemical conjugation and immunological evaluation of different neoglycoconjugates.
Synthesis of a library of glycans from Acinetobacter baumannii ATCC 19606 lipopolysaccharide core region.
1 • Prof. Barbara Richichi and Prof. Marco Marradi, Florence (University of Florence, I) to be trained in the conjugation of oligosaccharides to protein and gold nanoparticles;
2 • Prof. Thomas Vorup-Jensen, Aarhus (Aarhus University, DK) to be trained in the study of the immuno-toxicological profile of glycoconjugates.
MPICI has extremely well equipped laboratories, especially within the Biomolecular Systems department. The labs with 60 fume hoods include a walk-in fumehood for large set-ups, dry solvent systems, flow reactor devices (both commercial and built in-house) and flow-compatible benchtop NMR, FlowIR. Analytical techniques: NMR, HRMS, LC-MS, IR, UV, electron and atomic force microscopes. The team uses 5 automated glycan assembly systems (3 home built, two commercial systems) and has access to five NMR spectrometers (700 MHz, 600 MHz, 500 MHZ, 2x 400 MHz) MALDI and HRMS, ten HPLC systems and all other equipment needed to perform chemistry. In addition, the department maintains S1 and S2 biological facilities to work with bacteria and an animal facility to work with mice and rats also in a S2 environment.
The chemical synthesis of complex carbohydrates is a challenging process that requires multistep procedures and time-consuming purification of intermediates. As a result, the final product is usually obtained in low overall yield after many months to years of laborious work. GlycoUniverse (GU), in collaboration with Max-Planck-Institute (MPG), develops innovative Automated Glycan Assembly (AGA) technologies that enable autonomous production of complex oligosaccharides in several weeks. The primary tasks of the recruited DC will be 1) to identify optimal protective group strategies based on preparation of key monosaccharide building blocks, and 2) automated production of oligosaccharides of interest having various types of labels for biophysical studies. The synthetic compounds will then be tested for identity and functionality with the consortium’s partners during the recruited DC’s secondments. In addition, the recruited DC will have exposure to day-to-day operation of a vibrant international team of carbohydrate biochemist and automation experts.
Synthesis of oligosaccharide structures from Acinetobacter baumannii ATCC 17978, ATCC 17961 and ATCC 19606 polysaccharides larger than one repeating unit.
1 • Prof. Peter H. Seeberger, Potsdam (Max Planck Institute of Colloids and Interfaces, D) to be trained in cutting-edge NMR technology in carbohydrate structural analysis;
2 • Dr. Andrei Turtoi, Montpellier (Institut National de la Santé et de la Recherche Médicale – INSERM, F) to be trained in the generation of monoclonal antibodies using phage display methodology.
GlycoUniverse is currently located on the campus of the Postdam Science Park just outside Berlin in one of several Incubators called GO:IN where they
rent four fully equipped laboratories as well as office space. With a collaborative contract between GU and MPIKG established, GU has full access to MPIKGs analytical services such as NMR, HPLC, mass spectrometry, and Glyconeer automated synthesizers.
The rational design of glycoconjugate vaccines capable of conferring protection against the selected Acinetobacter baumannii (Ab) species may require an increasing number of oligosaccharides to be included in the vaccine formulation. The diverse glycan structures from different Ab strains generated by the consortium will be conjugated to different carriers capable of displaying saccharide fragments targeting the same pathogen, or a combination of oligosaccharide antigens related to distinct Ab strains, to create innovative multicomponent constructs. In this project supramolecular polymeric nanoparticles (NPs) will be used as multivalent nanoplatforms. Polymeric NPs will be produced by self-assembly of polyethylen glycol(PEG)ylated polyallylamine chains through amine phosphate interactions, which result in NPs highly homogeneous in size, that can be varied from 10 nm to a couple of hundreds of nm. The recruited DC will work on preparative aspects of NPs for vaccine formulation, exploring the synthesis of supramolecular NPs with a controllable density of glycans. The DC will conduct studies of the interaction of the NPs with protein conjugates complementary to the glycans on the NPs by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross Correlation Spectroscopy (FCCS). Other NPs synthesised by the consortium’s partners will be tested with the same techniques for comparison. In addition, the recruited fellow will perform pharmacokinetic studies to assess the biodistribution and translocation of the NPs following systemic delivery in mouse models.
Preparation and characterisation of supramolecular polymeric nanoparticles displaying a controlled density of glycan moieties on their surface in order to 1) study their interaction with proteins, and 2) assess their biological fate in vivo.
1 • Prof. Luigi Lay, Milan (University of Milan, I) to be trained in the synthesis of oligosaccharides and their conjugation to nanoparticles;
2 • Dr. Ahmed Ibrahim, Stockholm (Attana AB, S) to study the interaction of glycan-functionalized nanoparticles with lectins by Crystal Quartz Microbalance (QCM), and to compare the results obtained by FCS/FCCS using fluorescently labelled nanoparticles and lectins.
CIC biomaGUNE counts on a unique research infrastructure equipped with the most advanced nanoscience, biochemistry and molecular imaging facilities, including fully equipped research laboratories, Technological Platforms and the Molecular Imaging Facility, one of the biggest preclinical imaging research infrastructures in Europe. Characterization techniques for nanomaterials laboratories equipped with Transmission Electron Microscopy, scanning electron microscopy, Dynamic Light Scattering, Atomic Force Microscopy, Infrared spectroscopy, Raman spectroscopy, Confocal Laser Scanning Microscopy, Fluorescence Correlation Spectroscopy, Mass Spectroscopy facility. Facilities for in vivo imaging (PET/SPECT/MRI). Cell culture labs and animal facility. Synthetic laboratories State of the art laboratories for the synthesis of nanomaterials and polymers
Applications for the DC7 individual projects (Aarhus University) shall be submitted exclusively via the job portal of the Faculty of Medicine (Health) of the Aarhus University, where these two positions will also be advertised:
https://health.au.dk/en/about-health/vacant-positions
Applications for the DC7 projects submitted through the ACINETWORK website will not be considered.
It is key to establish that the glyconanoparticles (GNPs) generated by the consortium do not lead to an immune-tox response and determine how the different GNPs interacts with leucocytes, in vitro (cell lines) and in vivo (mice). The recruited DC will address these questions with three distinct and complementary approaches. First, the complement activation of the various GNPs will be investigated. To get a comprehensive overview of the properties, the activation will be followed in serum from several donors with assays highly relevant for understanding immune-toxicological responses. Deposition of relevant molecules on particle surfaces will be monitored with ELISA-like assays. As a biochemical supplement, the binding by vaccine GNPs to surfaces coated with plasma-captured IgM or recombinant human MBL will be analyzed. This will enable quantitative insights into the interaction of GNPs with the immune system. The second objective is to understand the interaction of naked or human serum-incubated GNPs with leukocytic cell lines (e.g., THP-1) or primary human monocytes. Using fluorescent GNPs, phagocytosis will be studied efficiently with image stream flow cytometry. Stains for phagolysosomal activity and/or ROS formation will be applied. The assays will also be modified to permit the study of cytokine production, especially the proinflammatory interleukins. Finally, it will be necessary to characterize some of the findings from the experiments described above. Since LD-50 determination for the GNPs is unlikely to meet ethical approval, complement activation will serve as a proxy measure of immune-tox properties. Likewise, the release of fibrinolytic peptides will be a proxy for intravascular coagulation. Fluorescent GNPs will be followed in instruments such as the In vivo Imaging System to determine the biodistribution. A focus will also be placed on key organs for myeloid phagocytes, namely the liver and spleen.
Comprehensive characterisation of the immuno-toxicological response of the glycosylated nanoparticles.
1 • Prof. Peter H. Seeberger, Potsdam (Max Planck Institute of Colloids and Interfaces, D) to be trained in synthesis of oligosaccharides and cutting-edge NMR technology for their structural analysis;
2 • Dr. Sergio E. Moya, San Sebastian (CICbiomaGUNE, E) to study the biodistribution and biological fate of polymeric and gold glyconanoparticles.
Technical expertise in: cell culture, western blot, RT-PCR, enzymatic assays, fluorescence and confocal microscopy approaches and electron microscopy techniques. The laboratory possesses a cell culture room and all equipment for daily biochemical and cell biological experiments, including a fluorescence microscope and equipment for the preparation and processing of electron microscopy samples.
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ACINETWORK will develop a battery of glycoconjugate vaccines, which differ in carrier, ligand composition (mono or bivalent) and ligand density. In vitro experiments in cell lines are key to determine which of them will be further examined for their vaccination potential in costly and time-consuming in vivo experiments. Glycoconjugate vaccines can elicit an immune response by either binding to the surface or internalisation through endocytosis and subsequent presentation on MHC class 2 molecules upon processing in the endolysosomal system. Thus, it is important to determine which glycoconjugate vaccines bind to the surface of a cell and/or are internalised, and whether they trigger an immune response. It is also relevant to discard those glycoconjugate vaccines that induce additional undesired cellular responses, as well as to determine whether the glycoconjugate vaccines are turned over after a certain time, an important aspect for biosafety. The major tasks of the recruited DC will be to 1) Determine the cell entry mechanism and the intracellular site(s) of accumulation of each developed glycoconjugate vaccine in the analysed cell types (internalisation experiments), 2) establish whether and how glycoconjugate vaccines are degraded intracellularly in the analysed cell type (biodegradability experiments) and 3) understand whether glycoconjugate vaccines entail an immune response (immunity experiments).
To gain insight into the mechanism of cell entry, intracellular fate and type of the immune response raised by glycoconjugate vaccine candidates against Acinetobacter baumannii.
1 • Dr. Andrei Turtoi, Montpellier (Institut National de la Santé et de la Recherche Médicale – INSERM, F) to be trained in the generation of monoclonal antibodies using phage display methodology;
2 • Dr. Roberto Testi, Rome (Fratagene Therapeutics, I) to be trained in drug repurposing strategies and their application on antimicrobial resistant bacteria.
Technical expertise in: cell culture, western blot, RT-PCR, enzymatic assays, fluorescence and confocal microscopy approaches and electron microscopy techniques. The laboratory possesses a cell culture room and all equipment for daily biochemical and cell biological experiments, including a fluorescence microscope and equipment for the preparation and processing of electron microscopy samples.
Successful vaccination against Acinetobacter baumannii (Ab) requires a set of innovations in the classical glycan-carrier protein immunization approach. One of the most relevant steps in this process, the design of the glycan portion of the complex, is limited by the knowledge of the exact glycoepitope. An elegant approach to determine this epitope consist of using a panel of antibodies to block the recognition between the glycan-bearing peptide and the cells of the immune system. However, raising antibodies against the glycan structure using immunisation is a challenging process as natural immunity is rather inefficient at this task. Therefore, a synthetic antibody-engineering approach, such as phage-display, bares more promise as it circumvents the need to properly activate various cellular immune components. In the DC9 Individual research project, the fellow will focus on raising monoclonal antibodies against the synthetic Ab glycoepitopes available from the consortium. For this purpose, an in-house available synthetic antibody library will be used, as well as an additional immune antibody library will be created from patients infected with Ab. Antibodies selected for their best binding and functional abilities will be further reformatted in full IgG, in order to achieve the best format for experimental studies as well as suitable nM affinities. Efficacies of the newly synthetized antibodies will be then functionally studied both in vitro as well as in vivo.
Generation of a set of monoclonal antibodies against synthetic Acinetobacter baumannii glycoepitopes using phage-display methodology, and assessment of their functional activity.
1 • Dr. Antonio J. Martín Galiano, Madrid (Instituto de Salud CARLOS III, E) to be trained in isolation of B cells from patients infected with Acinetobacter baumannii;
2 • Dr. Ahmed Ibrahim, Stockholm (Attana AB, S) to be trained in Crystal Quartz Microbalance (QCM) technique and test the affinity of selected antibodies.
INSERM Tumor Microenvironment and Therapy Resistance Lab has its main technical expertise in biomarker discovery (mass spectrometry, e.g. proteomics/ metabolomics), antibody engineering (phage display), ex vivo models (CAM model and tissue culture) & experimental pathology (histology and immunohistochemistry). We also operate a dedicated mass spectrometry facility with expertise in small molecule and metabolomics analysis.
Vaccination is a promising alternative to control intrahospital antibiotic-recalcitrant species such as Acinetobacter baumannii (Ab). As an extracellular pathogen, vaccine antigen candidates are expected to be surface-exposed and induce a strong adaptive humoral response. To evaluate this response, a strict assessment of the amount and variety of the specific raised antibodies is required. Moreover, effective candidates must elicit an effective protective response in animal models. Protection should ideally cover at least the principal high-risk Ab clones. The utilization of synthetic glycans from the capsular polysaccharides and LPS to reach immunoprotection is a promising alternative to other initiatives that involve surface proteins. The essential tasks of the recruited DC will be: 1) Evaluation of IgG levels and avidity induced by glycoconjugates in mouse serum samples by ELISA and surface plasmon resonance. 2) Determination of the immunoprotection (with and without adjuvant) by glycoconjugates against Ab lethal sepsis challenge using a mouse model. 3) Evaluation of the strain coverage of the oligosaccharide-based vaccine, in particular for antibiotic-resistant clones, using whole-cell ELISAs and genome-based computational tools.
Characterisation of the antibody response upon immunisation with Acinetobacter baumannii-derived glycoconjugates and the ability to induce protective immunity against multidrug resistant Acinetobacter baumannii strains.
1 • Prof. Fulvio M. Reggiori and Prof. Thomas Vorup-Jensen, Aarhus (Aarhus University, DK) to be trained in cell microbiology and use of advanced technologies to assess intracellular biodegradation of glycoconjugates, as well as in the study of the immune-toxicological profile of glycoconjugates;
2 • Dr. Laura Polito, Milan (National Research Council, SCITEC Institute, I) to be trained in the microfluidic production of functionalized nanomaterials.
Evaluation of IgG levels and avidity in mouse serum samples by ELISA and surface plasmon resonance; Determination of the immunoprotection (with and without adjuvant) against an Acinetobacter baumannii lethal sepsis using a mouse model; Evaluation of the strain coverage of the oligosaccharide-based vaccine, in particular, for antibiotic-resistant clones using whole-cell ELISAs and genome-based computational tools.
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Marie Skłodowska Curie Network
ACINetwork (Project no. 101119795)
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