Comparative proteomics identified immune response proteins involved in response to vaccination with heat-inactivated Mycobacterium bovis and mycobacterial challenge in cattle

•Evaluation of heat-inactivated M. bovis vaccine and mycobacterial challenge in cattle.•Comparative proteomics identified immune system pathways involved in vaccine response.•The most overrepresented immune proteins identified include TLR4, TLR9, C8α and C8β.•The JAK-STAT and PKC signalling pathways are potentially involved in vaccine response. There is an imperative need for effective control of bovine tuberculosis (BTB) on a global scale and vaccination of cattle may prove to be pivotal in achieving this. The oral and parenteral use of a heat-inactivated Mycobacterium bovis (M. bovis) vaccine has previously been found to confer partial protection against BTB in several species. A role for complement factor C3 has been suggested in wild boar, but the exact mechanism by which this vaccine provides protection remains unclear. In the present study, a quantitative proteomics approach was used to analyze the white blood cell proteome of vaccinated cattle in comparison to unvaccinated controls, prior (T0) and in response to vaccination, skin test and challenge (T9 and T12). The Fisher’s exact test was used to compare the proportion of positive reactors to standard immunological assays for BTB (the BOVIGAM® assay, IDEXX TB ELISA and skin test) between the vaccinated and control groups. Using reverse-phase liquid-chromatography tandem mass spectrometry (RP-LC-MS/MS), a total of 12,346 proteins were identified with at least two peptides per protein and the Chi2-test (P = 0.05) determined 1,222 to be differentially represented at the key time point comparisons. Gene ontology (GO) analysis was performed in order to determine the biological processes (BPs), molecular functions (MFs) and cell components (CCs) the proteins formed part of. The analysis was focused on immune system BPs, specifically. GO analysis revealed that the most overrepresented proteins in immune system BPs, were kinase activity and receptor activity molecular functions and extracellular, Golgi apparatus and endosome cell components and included complement factor C8α and C8β as well as toll-like receptors 4 (TLR4) and 9 (TLR9). Proteins of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) (JAK-STAT) and protein kinase C (PKC) signaling pathways were furthermore found to potentially be involved in the immune response elicited by the inactivated vaccine. In conclusion, this study provides a first indication of the role of several immune system pathways in response to the