Selection of relevant papers (criteria of inclusion), analysis of results of experimental infections describing immune responses, with and without the use of Brucellin: i) identification of immunogenic proteins; ii) cellular activation upon Brucella infection; iii) in vivo experimental models for immune response investigation; iv) identification of biomarkers of Brucella infection in experimental and production animals; v) collection of structural information on proteins that stimulate immune response. 

This WP will compare compositions of various commercial and in house Brucellin formulations and batches. Results will be used to determine the best formulation to organise in vivo experiments in WP3 and to develop ex vivo stimulation model in WP4. Following analyses will be performed: i) batch potency of various Brucellin formulations will be analyzed according to WOAH recommendations in guinea pig model; ii) ultrasensitive shotgun protein characterization of Brucellin formulations; iii) based on these results and literature data in silico analyses to identify the most likely immunogenic proteins and afterwards structural characterization using X-ray crystallography, cryo electron microscopy, and small-angle X-ray scattering will be used; iv) on these proteins, the structure-based in silico modelling of protein dynamics and antigen-receptor interactions will be applied 

This WP will investigate immune response markers through integrated proteo-genomics and meta-proteomic approaches. High-throughput “single cell” proteomic retrospective analyses of blood from different categories of animals (infected, vaccinated, FPSRs and brucellosis free) will be performed to identify individual cellular proteomes and understand the variability of markers at the best sensitivity possible. Metaproteomics will be performed with interpretation pipeline to proteotype the different organisms present in the samples and establish a clear pathological context for each sample. This will establish the serum markers of cellular response and prioritize the best candidates to be tested in WP4. Guinea pigs will be experimentally infected with smooth Brucella spp., and the cellular response will be monitored via blood samples in several time points. Guinea pigs will then be inoculated with Brucellin (from WP2), to analyse immune stimulation patterns (antibodies, cytokines, peptides, proteins). These results will be used to identify potential biomarkers for development of innovative methods, such as ex vivo stimulation, in WP4. 

Based on detection of biomarkers identified in WP2 and WP3, we will aim to develop an alternative diagnostic tool(s) for indirect brucellosis diagnostic, in sera from different categories of production animals (infected, vaccinated, FPSRs and brucellosis free). The evaluation of ex vivo stimulation model and follow up of IFN-gamma and other biomarkers in domestic species using available Brucellin formulations or new candidates as diagnostic tool will be performed. 

This WP is dedicated to the general coordination and management, including all administrative and financial issues, for timely dissemination of outputs. The coordinator will ensure successful management, integration and delivery of the project. Annual workshops and bi-monthly meetings will be organized to exchange information about the workplan and deliverables. A data management plan will be drafted to identify information generated by the project. The partners will be responsible for sharing newly generated data. All data, derivative analyses and protocols will be made available to the wider scientific community through repositories that follow the FAIR principles. The consortium will disseminate the knowledge in conferences and peer- reviewed journals, ensuring open access. The WP5 will assure integration of results through proposition of: i) new standardization schemes to control the Brucellin batches and lower the number of laboratory animals used; ii) refined schemes for brucellosis monitoring with the new diagnostic tools, to limit consequences of brucellosis management practices on animal welfare. 

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This work was co-funded by the European Union's Horizon Europe Project 101136346 EUPAHW. Views and opinions expressed are those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.