New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

Abstract

cThe tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test

“…Interestingly, immunological studies using antigen-displaying beads revealed that the beads showed adjuvant properties by enhancing the immune response to the displayed antigen, compared to its soluble counterpart (18). This paper extends findings from the earlier study (11) by demonstrating that biobeads displaying ESAT-6, CFP10, and Rv3615c (3-protein biobeads) were effective in identifying experimentally and naturally M. bovis-infected cattle, as well as distinguishing them from BCG-vaccinated noninfected animals. In addition, biobeads were designed and produced to display four mycobacterial proteins, i.e., ESAT-6, CFP10, Rv3615c, and Rv3020c (4-protein biobeads) simultaneously, potentially increasing test sensitivity (10).…”

“…As the skin test is the primary screening test for TB diagnosis in cattle, the cost of the skin test reagents is an important consideration. It was reported recently that display of three specific mycobacterial proteins (ESAT-6, CFP10, and Rv3615c) on bacteria-produced polyester inclusions (biobeads) could greatly increase their immunogenicity for the skin test (11). This markedly reduces the cost of the reagents, as lower concentrations of the proteins can be used and only a single fermentation is required with the proteins displayed as a fusion protein on single biobeads.…”

“…Polyester beads displaying three mycobacterial proteins, i.e., ESAT-6, CFP10, and Rv3615c (3-protein biobeads), were produced in E. coli BL21(DE3) as described previously (11). In order to produce polyester beads that simultaneously displayed four mycobacterial antigens, i.e., ESAT-6, CFP10, Rv3615c, and Rv3020c (4-protein biobeads), we designed and constructed a hybrid gene encoding all four antigens fused to a polyester synthase as a single polypeptide.…”

“…This has been achieved by displaying ESAT-6, CFP10, and Rv3615c on polyester inclusions (biobeads) produced by Escherichia coli, and preliminary results have indicated their utility in skin testing for the diagnosis of bovine TB (11). Polyester inclusions are naturally produced by various bacteria during imbalanced nutrient availability in which excess carbon is available and is deposited as spherical water-insoluble cytoplasmic inclusions (12,13).…”

Immunity and Vaccination against Tuberculosis in Cattle

“…A potential problem with the use of these antigens in a primary screening skin test would be the cost of the reagents. A recent study has shown that these antigens could be displayed on polyester inclusion bodies produced in bacteria such as Escherichia coli (Biobeads), producing a skin test reagent with a high sensitivity and specificity [84]. The cost of production of these Biobeads displaying a fusion protein of these three proteins is markedly lower than that of recombinant proteins or peptides as the antigens are used at very low concentrations due to their high immunogenicity and they are produced by fermentation technology.…”