Production of <b><i>Mycobacterium bovis</i></b> Antigens Included in Recombinant Occlusion Bodies of Baculovirus

Abstract

Bovine tuberculosis (bTB) is a disease produced by Mycobacterium bovis that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of M. bovis (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used M. bovis antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from Escherichia coli, because this bacterium produces a high level of recombinant proteins. However, E. coli recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c M. bovis antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same M. bovis proteins produced in E. coli.

Evaluating the viral clearance ability of continuous monoclonal antibody purification steps, in order to inactivate and/or remove four model viruses

“…In an IFN-γ assay, purified protein derivative of M. bovis fusion proteins showed equivalent sensibility but better specificity than the same M. bovis proteins produced in E. coli ( 19 ).…”

An ambient-temperature stable nanoparticle-based vaccine for nasal application that confers long-lasting immunogenicity to carried antigens

“…The fusion of antigens from baculovirus to the occlusion bodies have also been attempted. Several antigens such as herpesvirus II ( 13 ), Mycobacterium bovis ( 14 ), porcine circovirus type 2 ( 15 ), foot-and-mouth disease virus ( 16 ), and many others ( 17 ) have been fused to the wild type polyhedrin gene to generate recombinant occlusion bodies. Mice immunized with the recombinant polyhedras produced antibodies that recognize the pathogen, and in some studies prevent the disease ( 15 , 18 ).…”