ANSES will participate in WP1 - literature selection, analysis and data mining necessary to identify knowledge gaps, immunogenic intracellular protein candidates and organise further WPs. Within WP2, our team will perform Brucellin batch potency in guinea pig models according to WOAH recommendations and collect samples from these validation assays. For WP3, our team will prepare selection of production animals' sera for retrospective high-throughput proteomic analysis. Anses will participate in proteomic, peptidomic and meta proteomic data analysis, and will perform guinea pig in vivo studies. Within WP4, ANSES team will participate in development of ex vivo diagnostic tools and their validation on sera from negative and FPSR animals. As a project coordinator, ANSES assure general coordination and management, to ensure the timely implementation of the tasks, and successful dissemination of outputs. 

Our team will contribute to WP1 in selection of relevant literature and search for data relevant to this project. In WP3, with our vast bank of animal sera, we can contribute to retrospective analysis of production animals’ samples from Europe, Asia and Africa. These samples will give variability of pathogenic context to the analysis, since the collection constitutes various epidemiological situations. On sera from positive, false positive and serologically negative animals we can validate performances, such as sensitivity and specificity, of newly developed tools in WP4. Together with other colleagues within WP5, we will work on implementation of project results in surveillance strategies and decision-making regarding brucellosis management. Our implication and the use of animal sera from various epidemiological contexts will show the economical and sanitary benefits of improved policies and management practices. 

INIAV team will contribute to WP1 working on the literature review on brucellin and immunogenic proteins involved in Brucella infection and vaccination. As team leader, we will collaborate with all partners in disseminating updated knowledge; in WP3, as INIAV have a vast collection of infected-, vaccinated- and brucellosis-free sera, we will perform retrospective analyses of those sera. We will use Nanopore sequencing approach to profiling protein expression patterns in complex biological samples like serum. Additionally, we will explore the feasibility of incorporating methylation analysis techniques alongside nanopore sequencing to gain a deeper understanding of the epigenetic regulation involved in brucellin production; INIAV team will collaborate with other partners in the validation and implementation of newly developed tools in WP4. Regarding WP5, we will support the project management in the implementation of the data management plan, and in the dissemination of results. 

Ultra-sensitive proteomics analyses of different brucellin formulation, in-depth proteomics on guinea pig blood samples, deep-proteomics of sera from different categories of animals (infected, vaccinated, false positive serological reactions, brucellosis free), identification and prioritization of biomarkers of interest. 

Based on detection of bio-markers, identified in WP2 and WP3, we will aim to evaluate an alternative diagnostic tool(s) for indirect brucellosis diagnostic in blood from different categories of production animals, in particular the evaluation of a in vivo stimulation model and follow up of IFN gamma and other bio-markers in domestic species using commercially available Brucellin formulations or new candidates will be assessed as diagnostic tool. 

CNR-IC will provide the following contributions: 

WP1: review of current structural knowledge about key brucellin immunogenic proteins. Investigation on purification protocols and crystallization conditions used for crystal structure determination of their closest homologous. 

WP2: selection of best candidates for protein structure determination. Definition of the most appropriate strategies to achieve structural knowledge. Experimental and computational activities to achieve protein structural models. Computational modelling activities based on experimental structures to investigate protein dynamics and peptide-protein and protein-protein interactions. Identification of new immunogenic biomarkers. 

WP5: setting up the data management plan, participation in project meetings, promote cross-fertilization among partners, disseminate the results and contribute to developing IP strategies .

Sciensano will be involved in WP1, WP3, WP4 et WP5. 

IZS-Teramo contribution: WP1: literature review and identification of key knowledge information on brucellin and immune response to Brucella infection and vaccination. WP2: proteomic analyses of brucellin antigens and in silico analyses for single protein characterization and protein-protein interactions for identification of immunogenic targets. IZS-Teramo will contribute to perform brucellin batch potency in the guinea pig model WP3: provision of positive sera from brucella vaccinated and infected animals for identification of signature biomarker and characterization of immune response of experimentally infected guinea pigs using multiparametric luminex technology WP4: development of new assay based on whole blood ex-vivo stimulation with brucellin antigen and quantification of signature marker of cell-mediated immune response (e.g. IFN-gamma) WP5: support to project management and implementation and dissemination of project results.