CONICET | Buscador de Institutos y Recursos Humanos

INTRODUCTIONPhoP and Mce2B proteins are two virulence factors in mycobacteria involved in transcriptional regulation and macromolecules transport, respectively. Two vaccine candidates, M. bovis Δmce2 and M. bovis Δmce2ΔphoP, have been designed based on the deletion of coding genes for these proteins by gene. A DIVA type diagnostic system compatible with the potential use of these two experimental M. bovis vaccine candidates should be developed, capable of differentiating vaccinated from infected animals. Recombinant PhoP (rPhoP) was described as humoral antigen in human tuberculosis, but there is no evidence about the antigenic properties of recombinant Mce2B(rMce2B) in the context of M. bovis infection. In the present study, we evaluated the cell-mediated immune response against rPhoP and rMce2B proteins in naturally infected (TBC) and non-infected (non-TBC) cattle.MATERIALS AND METHODSThe recombinant vectorspET-15b-phoP (kindly provided by Dr.Jesus Gonzalo Asensio,Zaragoza University)andPet28-Mce2B were expressed in th competent Escherichia coli BL21pLYs strain inLBmedia,ampicillin(100ug/ml)andIPTG(0.1mM).Inclusion bodies were recovered as previously described(4)and recombinant proteins were purified by an agarose-nickel affinity chromatography(ProteinIsoNi-NTAResin,TransGenBiotechCo.,Ltd.)under denaturing conditions (buffer:8M urea, 20mM sodium phosphate and 500mM NaCl, pH4.5).Eluates were visualized on a15%SDS-page with colloidal coomassie staining and by Western blot(Primary antibody: Mouse Mab Anti-polyHistidine H1029,Sigma-Aldrich. Secondary antibody: alkaline phosphatase-conjugated antimouseimmunoglobulinG,Sigma).For detection,BCIP/NBTcolor development substrates (Promega, Madison,WI)were used. PhoPr and Mce2Br werec onfirmed by Orbitrap technology (ThermoScientific,Q-Exactive). The predicted molecular weight of PhoP and Mce2B was performed using ExPASy(5).Heparinised blood from10non-TBC, 6 anergic with visible tuberculous lesions (VTL) and 7TBC animals (positive to tuberculin test and/or IFN-gamma test) were obtained. Blood was stimulated with rPhoPandrMce2B, individually (10µg/mL final concentration),are combinant fusion protein (FP)3615c/ESAT6/CFP10(10ug/mL) (kindly provided by Dr. Martin Vordermeier), avian and bovine tuberculin (PPDA and PPDB,respectively),PBS1X and acellular mitogen (Pokeweed, PKW). BovigamKit (Thermofisher) was used to measure IFN gamma in supernatants of stimulated blood. A threshold=0.1 was considered. Kruskal Wallis test and a Dunn`spost-test were performed to identify a significant response.ResultsTBC animals: The magnitude of the response was similar between them (p=0.124), and in the case of rMce2B and rPhoPless than PPDB (p=0.0083). An overlapping response was observed among rMce2B, rPhoPand the FP, except for a TBC animal that was only reactive to rMce2B and rPhoP. Anergic animals: The magnitude of the response was similar between them, including the PPDB antigen (p=0.1989). An overlapping response was observed between rMce2B, rPhoPand FP, except for one anergic animal that was only reactive to rPhoP.ConclusionsVirulence related proteins are attractive molecules to be proven as diagnostic antigens in infectious diseases. Some of them such as ESAT6, CFP10, 3615c have been widely characterized and used as specific antigens in bovine tuberculosis diagnosis. In the present study, rPhoPand rMce2B from M. boviswere assayed showing they induced cell-mediated immune response although with a lower responder frequency than the FP and PPDB in naturally infected cattle. However, this is a preliminary study that should be assayed in a larger sample size to further analyze these two experimental recombinant proteins as diagnostic antigens in the context of the potential use of M. bovisΔmce2 and M. bovisΔmce2ΔphoP vaccine candidates.