Protein Levels of Pro-inflammatory Cytokines and Chemokines as Biomarkers of Mycobacterium bovis Infection and BCG Vaccination in Cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a globally prevalent infectious disease with significant animal welfare and economic impact. Difficulties in implementing test-and-slaughter measures in low- and middle-income countries (LMICs) and the underperformance of the current diagnostics establish a clear need to develop improved diagnostics. Adaptive immunity biomarkers other than IFNγ could be useful as suggested by various gene expression studies; however, a comprehensive assessment at the protein level is lacking. Here, we screened a range of chemokines and cytokines for their potential as biomarkers in samples from M. bovis experimentally challenged or naive animals. Although serum concentrations for most proteins were low, the pro-inflammatory markers, IL-2, CXCL-9, IP-10 and CCL4, in addition to IFNγ, were found to be significantly elevated in bovine tuberculin (PPDb)-stimulated whole blood supernatants. Further assessment of these molecules in BCG-vaccinated with or without subsequent M. bovis challenge or naive animals revealed that PPDb-specific IL-2 and IP-10, in addition to IFNγ, could discriminate naive and BCG-vaccinated from M. bovis challenged animals. Moreover, these proteins, along with CCL4, showed DIVA potential, i.e., enabling differentiation of M. bovis-infected animals from BCG-vaccinated animals. Combined analysis of cytokines and chemokines could also accurately identify M. bovis infection with strong correlations observed between PPDb-specific IFNγ, IL-2 and IP-10 levels. This provides proof of concept for utilizing multiple biomarker signatures for discrimination of animals with respect to M. bovis infection or BCG vaccination status

Improved Prediction of Bovine Leucocyte Antigens (BoLA) Presented Ligands by Use of Mass-Spectrometry-Determined Ligand and in Vitro Binding Data

Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predictors of peptide-MHC interactions constitute an attractive alternative. Recently, an increasing amount of MHC presented peptides identified by mass spectrometry (MS ligands) has been published. Handling and interpretation of MS ligand data is, in general, challenging due to the polyspecificity nature of the data. We here outline a general pipeline for dealing with this challenge and accurately annotate ligands to the relevant MHC-I molecule they were eluted from by use of GibbsClustering and binding motif information inferred from in silico models. We illustrate the approach here in the context of MHC-I molecules (BoLA) of cattle. Next, we demonstrate how such annotated BoLA MS ligand data can readily be integrated with in vitro binding affinity data in a prediction model with very high and unprecedented performance for identification of BoLA-I restricted T-cell epitopes. The prediction model is freely available at http://www.cbs.dtu.dk/services/NetMHCpan/NetBoLApan . The approach has here been applied to the BoLA-I system, but the pipeline is readily applicable to MHC systems in other species

Anti-parasitic benzoxaboroles are ineffective against Theileria parva in vitro

East Coast Fever (ECF) is a disease affecting cattle in sub-Saharan Africa, caused by the tick-borne Apicomplexan pathogen Theileria parva. The disease is a major problem for cattle farmers in affected regions and there are few methods of control, including a complex infection and treatment vaccine, expensive chemotherapy, and the more widespread tick control through acaricides. New intervention strategies are, therefore, sorely needed. Benzoxaboroles are a versatile class of boron-heterocyclic compounds with demonstrable pharmacological activity against a diverse group of pathogens, including those related to T. parva. In this study, the in vitro efficacy of three benzoxaboroles against the intracellular schizont stage of T. parva was investigated using a flow cytometry approach. Of the benzoxaboroles tested, only one showed any potency, albeit only at high concentrations, even though there is high protein sequence similarity in the CPSF3 protein target compared to other protozoan pathogen species. This finding suggests that benzoxaboroles currently of interest for the treatment of African animal trypanosomiasis, toxoplasmosis, cryptosporidiosis and malaria may not be suitable for the treatment of ECF. We conclude that testing of further benzoxaborole compounds is needed to fully determine whether any lead compounds can be identified to target T. parva

Protection associated with a TB vaccine is linked to increased frequency of Ag85A-specific CD4⁺ T cells but no increase in avidity for Ag85A

AbstractThere is a need to improve the efficacy of Bacille Calmette-Guérin (BCG) vaccination against tuberculosis in humans and cattle. Previously, we found boosting BCG-primed cows with recombinant human type 5 adenovirus expressing antigen 85A (Ad5-85A) increased protection against Mycobacterium bovis infection compared to BCG vaccination alone. The aim of this study was to decipher aspects of the immune response associated with this enhanced protection. We compared BCG-primed Ad5-85A-boosted cattle with BCG-vaccinated cattle. Polyclonal CD4+ T cell libraries were generated from pre-boost and post-boost peripheral blood mononuclear cells – using a method adapted from Geiger et al. (2009) – and screened for antigen 85A (Ag85A) specificity. Ag85A-specific CD4+ T cell lines were analysed for their avidity for Ag85A and their Ag85A epitope specificity was defined. Boosting BCG with Ad5-85A increased the frequencies of post-boost Ag85A-specific CD4+ T cells which correlated with protection (reduced pathology). Boosting Ag85A-specific CD4+ T cell responses did not increase their avidity. The epitope specificity was variable between animals and we found no clear evidence for a post-boost epitope spreading. In conclusion, the protection associated with boosting BCG with Ad5-85A is linked with increased frequencies of Ag85A-specific CD4+ T cells without increasing avidity or widening of the Ag85A-specific CD4+ T cell repertoire