BRU-CELL-IN - Project results

Project results

GANTT CHART

MILESTONES

M1.1 Month4 Identification of relevant papers

Critical identification of relevant peer review papers will be performed by all partners. List of potential papers will be proposed by all participants and ANSES will provide full texts. Main criteria for paper inclusion and further analysis will be the detailed description of materials and methods as well as results.

M2.1 Month10 Shotgun brucellin analysis

The whole dataset of proteins and their quantities will be established for the different Brucellin formulations. These data will be formatted for the PRIDE international data repository.

M2.2 Month24 Achievement of new structure knowledge

The first structure of a protein selected among those present in Brucellin formulations will be deposited in public databases. It will come from MX, SAXS or cryo-EM measurements and data analyses performed in T2.5. The structure will be released in PDB, SASBDB or EMDB, respectively.

M3.1 Month24 Proteomes of stimulated guinea pigs

The whole dataset of proteins and their quantities will be established for the different samples from the stimulated infected guinea pigs. These data will be formatted for the PRIDE international data repository.

M4.1 Month28 Optimized brucellin formulations

Target proteins selected in T2.3 and structurally characterized in T2.6 will be optimized for antigen-receptor or antigen-antibody interactions in T4.?, to be used respectively to enhance the effectiveness of Brucellin formulations, or the sensitivity and selectivity of brucellosis detection. The release of the first protein designed and tested for one of these functions will represent an important project milestone.

DELIVERABLES

D1.1 Month 12 Literature review

Current knowledge, gap analysis and urgent needs in the field will be drafted in a literature review to resolve the problem of FPSRs.

D2.1 Month 20 Batch composition consistency

Since Brucellin is LPS-free mixture of Brucella intracellular proteins, the composition of different batches will be analyzed as well as protein concentrations and ratios. This will determine the variability of batches and their compound differences. A report describing how was performed the sample preparation, measurements by high-resolution tandem mass spectrometry, and data analysis.

D2.2 Month24 Structures by MX, SAXS or cryo-EM

A report describing sample preparation, data collection, data analysis and validation performed in T2.6 will include relevant parameters describing experimental data and model refinement, besides parameters characterizing the structure found, such as radius of gyration, maximum interparticle distance in case of SAXS data. A detailed inspection on active site, including plots of the electron density map superposed to the reconstructed all-atom model will be reported.

D2.3 Month20 Brucellin batch potency report

Using the latest shot-gun proteomic approach the batch compositions and protein ratios will be determined. This will be then compared to in vivo potency tests on guinea pigs to identify the prominent immunogenic components of Brucellin.

D3.1 Month20 Characterisation of guinea pigs immunity

To characterize the guinea pigs’ immune response against Brucellae commercially available kits for the measurement of antibody concentrations, 12 plex kit for pro- and anti- inflammatory cytokines as well as kit for proliferation of T cells. Furthermore, PMBCs will be isolated and their stimulation with inactivated Brucellae will be tested. The results will be compared with data available from literature to prepare peer reviewed publication and data will be available upon the request.

D3.2 Month26 Report on guinea pigs stimulation

PBMCSs and several lymph nodes will be isolated at the end of each experiment, immune response will be examined as well as stimulation patterns. A report describing how was performed the sample preparation, measurements by high-resolution tandem mass spectrometry, and data analysis, with data listing the identified proteins and their quantities in the different samples analyzed.

D3.3 Month18 PBMCs protocols

Protocol for isolation of peripheral blood mononuclear cells from experimental animals will be provided to obtain PBMCs from naturally infected, FSPRs and brucellosis negative production animals. This protocol will include the procedure and shipping conditions so that isolated cells can be transported in shortest possible time to partners’ laboratories for ex vivo stimulation assays.

D3.4 Month30 Field versus experimental PBMCs

The report on the cellular stimulation from experimental guinea pigs and field obtained production animals’ PBMCs will be compared in order to identify ideal markers of Brucella infection.

D4.1 Month22 Structure-based optimization report

A report will include results of computational activities carried out to design specific mutations and test their effect on structural stability of the protein, as well as experimental activities to test the performance of mutated proteins in enhancing the efficacy of the Brucellin formulation, or the sensitivity and selectivity of new diagnostic tools based on the mutated protein as biomarker.

D4.2 Month34 Ex vivo stimulation assay report

The report will include results of computational activities carried out to design specific mutations and test their effect on structural stability of the protein, as well as experimental activities to test the performance of mutated proteins in enhancing the efficacy of the Brucellin formulation, or the sensitivity and selectivity of new diagnostic tools based on the mutated protein as biomarker.