Jenner Institute, UK
Title: Evaluation of the effect of BCG immunisation on the efficacy of vaccine against malaria developed at the Jenner Institute, in a mouse model. Grantee: Brankica Filipić, Department of Microbiology and Immunology, Faculty of Pharmacy - University of Belgrade, Serbia. STSM Period: 29.09.2018 to 31.12.2018. Host: Dr Anita Miličić, The Jenner Institute, University of Oxford, Oxford, UK. Purpose. The aim was to examine the effect of BCG immunisation on the efficacy of a vaccine against malaria, developed at the Jenner Institute, in a mouse model of malaria. In order to examine the adjuvanting effect of BCG, main goals of this STSM were: 1) to asses the most potent BCG dose, and 2) to establish the best route of BCG immunization.
Description of the work. Work carried out can be divided in two main parts: gaining experience in this mouse malaria model and testing the effect of BCG vaccination. We tested an SSI stain of BCG using varying doses (4x105 – 106 CFU) of BCG and two administration routes - intradermal (in the ear) and intramuscular. Malaria challenge was performed by i.v. injection of P. berghei (1,000 sporozoites) 2 weeks post immunisation. The main results. Using the P. berghei mouse malaria challenge model, we tested the effects of BCG on enhancing the protective efficacy of a vaccine against malaria. The results showed a significant improvement in protection against against challenge with P. berghei, compared to no surviving mice in the malaria vaccine-only or BCG-only groups.
Department of Pharmacy, NORWAY
Title: Establishing of ELISA method to measure the immune response of rainbow trout to a bacterial vaccine. Grantee: Konstantina Bitchava, Laboratory of Fish, Shellfish and Crustacean Diseases, Veterinary Research Institute, Thermi, Greece. STSM Period: 19.11.2018 to 5.12.2018. Host: Tor Gjøen, Farmasøytisk institutt, Universitetet i Oslo. Purpose. The purpose of this STSM was to provide me with information and guidelines to perform ELISA on fish sera. The aim was to establish an ELISA method to measure the immune response of rainbow trout to a bacterial vaccine. It will serve as an important tool for the implementation of experimental trials on fish in order to evaluate the immune response of fish to different vaccine adjuvants and routes of vaccination. Description of the work. Upon my arrival to the laboratory I was trained in using the Plate Washer and Plate Reader and preparing all the relevant solutions for the ELISA tests. We started our trials using different dilutions of the positive and negative sera in order to establish the most appropriate. We also tested different dilutions of bacterin for coating the plates. Generally we tested all the relevant parameters to obtain the most appropriate protocol. The main results. I worked with samples taken from my laboratory, on development of ELISA assays for monitoring fish immune responses after vaccination. This ELISA protocol was established for a gram+ bacteria of rainbow trout, Lactococcus garviae, which is the most important pathogen of cultured rainbow trout in Greece. Future collaboration with host. This STSM fostered the existing research network and the collaboration between the School of Pharmacy, University of Oslo and the Veterinary Research Institute, Thessaloniki, Greece with both laboratories having a common interest on fish immunology and the development of vaccines able to help and promote the aquaculture sector.
Scientific Institute of Public Health Sciensano, Belgium
Provisional title: Understanding the practical aspects of the EU administrative procedure for Official Control Authority Batch Release (OCABR) and laboratory methodology for different types of adjuvanted vaccines. Grantee: Nemanja Turković, Agency for medicines and medical devices of Montenegro, Podgorica, Montenegro. STSM Period: from 04.02.2019 to 08.02.2019. Host: Lieke Van der Aa, service Quality of Vaccines and Blood Products (QVPS), Scientific Institute of Public Health Sciensano, Belgium. Purpose. The service QVPS is an Official Medicines Control Laboratory (OMCL) for biological medicinal products for human and veterinary use. Each batch of a biological product, whether human or veterinary, must be controlled by an OMCL (in Belgium or any other Member State of the EU) before it is placed on the market. The batch must comply with the approved specifications provided for in the monographs of the European Pharmacopoeia and the marketing authorisation file concerned. The OMCL then issues a batch release certificate to the manufacturer. The aim of the STSM was to understand the practical aspects of the EU administrative procedure for Official Control Authority Batch Release (OCABR) and laboratory methodology for different types of adjuvanted vaccines. Collecting, explanation and understanding guidelines/regulations on the use of adjuvants for different groups of vaccines, understanding the traceability of the steps included in the procedure of batch release (issuing the OCABR certificates) and explanation of different in-house assays (ELISA, biochemical assays, in vivo assays) for vaccines with adjuvants were specific aims of this STSM. Description of the work. The main topics of the STSM were: (a) Explanation of manufacturing process, releasing testing by manufacturer and releasing testing by National Control
Laboratory; (b) Introduction and explanation of guidelines/regulations on the use of adjuvants for different groups of vaccines; (c) Introduction and explanations of dossier registration procedures of vaccines with adjuvants; (d) Presentation of specific products - vaccines with adjuvants (manufacturing process, quality control, presentation of testing, validation dossier and cases out of specification); (e) General introduction to EDQM and OMCL’s in the framework of batch release of vaccines with adjuvants (principle of batch release in Europe, batch release activities (QA and testing activities), quality surveillance of vaccines, methods transfer process); (f) Introduction and explanation of OCABR procedures and guidelines for vaccines with adjuvants, and (g) Explanation of different in-house assays (ELISA, biochemical assays, in vivo assays) + laboratory visits. The main results. The results can be summarized as following: (i) Collecting and understanding guidelines/regulations for different groups of vaccines; (ii) Collecting and understanding product-specific OCABR guidelines and procedures for vaccines with adjuvants; (iii) Understanding the traceability of the steps included in the procedure of batch release (issuing the OCABR certificates); (iv) Strengthening institutional capacities of CALIMS, and (v) Strengthening and expanding links among vaccine regulators, experts and different stakeholders. Previous mentioned results will contribute to improvement of system in Montenegro which ensures that only vaccines of assured quality are available for the population, in the interest of protection of public health and furthering research in this field of interest. Future collaboration with host. Discussions and knowledge exchange in the field of vaccines will be continued between Sciensano and CALIMS and new models of collaboration will be established.
Trinity College Dublin (TCD), Ireland
Title: Understanding dendritic cell and NLRP3 inflammasome activation by cationic liposomal adjuvants CAF01, CAF04 and CAF09. Grantee: Aneesh Thakur, University of Copenhagen (UCPH). STSM Period: 04.02.2019 to 01.03.2019. Host: Prof. Ed Lavelle, Trinity College Dublin (TCD), Ireland. Purpose. To investigate activation of dendritic cells (DC) and the NLRP3 inflammasome by cationic adjuvant formulation (CAF) adjuvants- CAF01, CAF04, CAF09, and CAF01-based adjuvants- PLGA-CAF01 and PLGA-CAF01-Chitosan. Description of the work. The role of specific signaling pathways mediating inflammasome activation and dendritic cell maturation was studied in mouse-originated bone marrow-derived dendritic cells (BMDCs) in the presence or absence of CAF-based liposomal formulations. The main results. We found that CAF-based formulations prepared with either PLGA and/or chitosan induce maturation of dendritic cells in a dose-dependent manner, where low doses of CAF i.e. 1.9/0.39 µg/ml of DDA/TDB induce DC maturation as compared to 15.6/3.125 µg/ml of DDA/TDB. We also found that all CAF or CAF01-based formulations tested in this study activate the inflammasome when combined with a TLR4 agonist. Further, PLGA and/or chitosan-based CAF01 formulations but not the CAF formulations induce IL-6 production by dendritic cells. Future collaboration with host. The grantee lab at UCPH and the host lab at TCD have common research interests in developing novel adjuvants. The data generated in this STSM will be further improved upon by conducting additional studies at UCPH in collaboration with the host lab. Future collaborative research proposals beyond this research project are also expected. Foreseen publications/articles. The data generated in this STSM project is novel and will be supported with additional studies planned at UCPH that includes studying the specific role of NLRP3 inflammasome, effect of caspase inhibition, and in vivo demonstration of the role of NLRP3 inflammasome. The results will be compiled and communicated for publication in a leading scientific journal.
Sciensano, Brussel, Belgium
Title: Training on different in vitro assays related to the assessment of inflammatory responses of dendritic cells (DC) and macrophages (Mf) to different stimuli. Grantee: Biljana Bufan, University of Belgrade – Faculty of Pharmacy, Belgrade, Serbia. STSM Period: 18.3.2019 to 29.3.2019. Host: Marta Romano, “In vivo models” unit, Scientific Service “Immune Response”, Scientific Directorate “Infectious diseases in humans”, Sciensano, Brussel, Belgium. Purpose. The purpose of this STSM was to get a training on the different in vitro assays that are related to assessment of inflammatory responses of dendritic cells (DC) and macrophages (Mf) to different stimuli. These assays could be useful for testing compounds with potential adjuvant activity. Also, signaling pathways that are used by testing compounds were evaluated. Description of the work. DC and Mf were generated in vitro from the precursor cells taken from the bone marrow of C57BL/6 wild-type (WT) or knock-out mice (Mincle-/- and MALT-1-/-). After generation, cells were activated and subjected to different assays to assess their inflammatory response. For activation, glycolipids with potential adjuvant activity: synthetic cis-alpha glucose monomycolate (GMMsynth) and hemi-synthetic glucose monomycolate (GMMnatmix) were used. Trehalose- 6,6-dibehenate (TDB) was used as control. The inflammatory response to tested compounds was assessed in DC by immunophenotypic determination of surface molecules with a role in T cell activation (CD11, CD11b, MHC II, CD80, CD86), and analysis of the production of reactive oxygen species (ROS) and TNF-α (pro-inflammatory cytokine), and in Mf by analysing the production of TNF-α. Immunophenotype and ROS levels were analysed by flow cytometry. TNF-α was determined in supernatants of cultures by sandwich ELISA assays. The main results. In DC from WT, all tested compounds induced similar inflammatory responses, according to performed assays. These results indicated inflammatory potential of tested compounds. DC and Mf from knock-out mice were used for revealing signaling pathways involved in action of the tested compounds. Mincle is a C-type lectin receptor and MALT-1 is adaptor protein involved in Mincle-NF-kB signaling pathway. Tested compounds did not induce inflammatory responses in DC and Mf from knock-out mice, according to the performed assays. Thus, usage of C-type lectin Mincle dependent mechanism in DC and Mf by tested compounds, in achieveing inflammatory effect, was suggested. Future collaboration with host. I hope acquiantances I acquired here and exchanged experiences will result in future collaborations in the field of discovery compounds with adjuvant activity.
Instituto di Ricovero e Cura a Carattere Scientifico per l’Oncologia, Genova, Italy.
Title: The immunomodulatory potential of Allostatine and the assessment of its toxicity and genotoxicity. Grantee: Imène ben Toumia, Research Unit Bioactive Natural Products and Biotechnology UR17ES49, Faculty of Dental Medicine of Monastir, University of Monastir, Tunisia. STSMs Period: 09.05.2019 to 09.08.2019 and 05.10.2019 to 05.01.2020. Host: Prof. Camillo Rosano, Instituto di Ricovero e Cura a Carattere Scientifico per l’Oncologia, Genova, Italy. Purpose. Allostatine is a peptide belonging to the Alloferon class and have been reported to be particularly perspective for adjuvant cancer immunotherapy. This study investigated the immunomodulatory potential of allostatine and the assessment of its toxicity and genotoxicity. Description of the work. We investigate the safety and the immunomodulatory potential of allostatine. The effects of allostatine on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells and we evaluated macrophage functions. In adition, we have assesed the toxicity and the genotoxicity of allostatine. The main results. We conclude that Allostatine may be potentially useful for modulating immune cell functions in physiological and pathological conditions and thus a good candidate as adjuvant cancer immunotherapy furthermore genotoxity test indicated that allostatine induced no toxic effects. Future collaboration with host. Our immunomodulatory project for allostatine supported by Short Term Scientific Missions (STSM) program shows promising results for adjuvant cancer immunotherapy vaccine. However, detailed Pharmacokinetics and ADMET of the new adjuvant must be performed. Foreseen publications/articles. Ex-vivo assay of Allostatine, a novel non toxic adjuvant cancer vaccine, that improves the immunomodulatory potential in C57BL6J mice.
Statens Serum Institut, Copenhagen, Denmark
Title: Evaluation of the immunogenicity and potential adjuvant properties of two developed thermosensitive hydrogels (ANOP1 and S97OP1) sublingually administered in mice. Grantee: Lorena García del Río, Dept. of Pharmacology - Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago, Santiago de Compostela, Spain. STSM Period: 30.09.2019 to 17.02.2020. Host: Statens Serum Institut, Copenhagen, Denmark. Purpose. The main purpose of this STSM was to evaluate the immunogenicity and potential adjuvant properties of two developed thermosensitive hydrogels, ANOP1 and S97OP1, when they were sublingually administered in mice. Description of the work. During the STSM period, two in vivo studies were carried out in mice. ELISA kits were used to measure mucosal and systemic immune responses in spleen, serum, cervical lymph nodes, vaginal wash and BAL. The main results. In general terms, we could conclude that for getting better immune responses a prime subcutaneous vaccination is required before mucosal boosting with hydrogels. Foreseen publications/articles. The results obtained have generated a potential joint publication.
University of Leiden, the Netherlands.
Title: In vitro investigations of adjuvant properties of selected novel PRR agonists. Grantee: Samo Guzelj, Faculty of Pharmacy, University of Ljubljana, Slovenia. STSM Period: 13.01.2020 to 31.01.2020. Host: Prof. Slütter, University of Leiden, the Netherlands. Purpose. As part of my PhD research, we developed a library of potent NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) agonists. Based on preliminary in vitro and in vivo screening, we chose the best performing compounds and covalently linked them to TLR7 (Toll-like receptor 7) agonists to produce chimeric NOD2/TLR7 dual agonists. As part of this STSM, we assessed the adjuvant capabilities of these chimeric conjugates by testing them on mouse BMDCs (Bone marrow derived dendritic cells), evaluating how the selected compounds influence antigen presentation to T‑lymphocytes and how that reflects on T cell activation and proliferation. Description of the work. BMDCs were stimulated overnight by selected compounds and ovalbumin as the antigen of choice, after which either CD4+ or CD8+ T-cells were added (isolated from spleens of OT-II or OT-I transgenic mice, respectively). After 3 days of co-culturing, the activation and proliferation of lymphocytes were assessed with flow cytometry. Additionally, concentrations of various cytokines in the supernatants were determined. The main results. The chimeric NOD2/TLR7 dual agonists proved to be capable of activating BMDCs, significantly enhancing antigen presentation of ovalbumin to T-lymphocytes even at concentrations as low as 10nM. The results were comparable to the LPS positive control in experiments with both CD4+ and CD8+ T-cells, showing that the compounds were capable of eliciting a balanced immune response stronger than individual NOD2 or TLR7 agonists or their mixtures, thus establishing conjugation of NOD2 and TLR7 agonists as a promising avenue in novel adjuvant development. Future collaboration with host. Since the development of novel adjuvants is an on-going process and the BMDC - T-Cell antigen presentation assay proved to be effective at evaluating the adjuvant potential of synthesized compounds, any further development will hopefully include prof. Slütters expertise and access to techniques otherwise unavailable at the Faculty of Pharmacy, University of Ljubljana. Foreseen publications/articles. Based on the promising results, the collaboration that was developed by the STSM will most likely lead to a joint publication between the research groups in the Faculty of Pharmacy, University of Ljubljana and Division of BioTherapeutics, University of Leiden. This publication will include both the results obtained during the STSM and data previously determined in the Faculty of Pharmacy, University of Ljubljana.
Rudjer Boskovic Institute, Zagreb, Croatia
Title: Introduction of techniques required for manipulation of the adenovirus vectors. Grantee: Pavle Banovic, Pasteur Institute Novi Sad, Serbia. STSM Period: 27.01.2020 to 10.02.2020. Host: Dragomira Majhen, Rudjer Boskovic Institute, Zagreb, Croatia. Purpose. The purpose of the STSM was to enhance international collaboration, share new techniques, and infrastructure that is now available in Grantee Institution. In addition, the general purpose of the STSM was to broaden my knowledge on adenoviruses as vectors for gene therapy and vaccination. Description of the work. The training was focused on laboratory techniques required for manipulation of adenovirus vectors. As part of the STSM, I have acquired knowledge on experimental in vitro methods – observation of adenovirus-induced cytopathogenic effect (CPE) on various cell lines, collection of adenovirus from cell cultures and concentration via centrifugation in gradient of caesium-chloride, and labeling of adenoviruses using AlexaFluor dye and purification with gel filtration. I have also perform passages of A 549 and Hek 293 cell lines and their clones, and got familiarised with their specificities. The main results. During this research we have gained knowledge about the interactions of adenovirus type 26 with αvβ3 Integrins on epithelial cell lines and mechanisms of adenovirus type 26 internalisation. This STSM refers to my future projects, where I plan to use acquired knowledge and experience in my home Institution. Future collaboration with host. The collaboration between Rudjer Boskovic Institute and Pasteur Institute Novi Sad has now been established. Both institutions are committed to achieve collaborative projects and to continue working together. Other comments. I would like to thank ENOVA for giving me this STSM and allowing me to expand my expertise and competencies.