Evaluation of Recombinant <i>Mycobacterium tuberculosis</i> Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs

Abstract

The tuberculin Purified Protein Derivative (PPD) skin test is widely used; however, the results are often inaccurate. Positive results can be observed in patients with active tuberculosis (TB) as well as in BCG-vaccinated persons and individuals who are infected with mycobacteria but have not developed the disease. MPT64, an antigen secreted from actively growing Mycobacterium tuberculosis and some strains of M. bovis BCG such as BCG Tokyo and BCG Russia, is immunogenic and elicits delayed-type hypersensitivity (DTH) in guinea pigs and humans. This antigen has been used to develop a new skin test for the diagnosis of active TB infection. Two of the antigens encoded by the M. tuberculosis-specific region of difference 1 (RD1, deleted in M. bovis BCG strains), CFP10 (culture filtrate protein 10) and ESAT6 (early secreted antigenic target-6), also induce M. tuberculosis-specific DTH responses. The aim of this study was to investigate the DTH responses in guinea pigs infected with M. tuberculosis or M. bovis BCG Tokyo elicited by three purified recombinant proteins (rMPT64, rCFP10 and rESAT6) compared to those elicited by PPD. In this study genes encoding MPT64, CFP10, and ESAT6 were cloned and expressed as recombinant proteins with the addition of a C-terminal His6 tag for ease of purification by Immobilized Metal ion Affinity Chromatography (IMAC). The recombinant proteins (rMPT64, rCFP10, and rESAT6) were purified to homogeneity and were used to elicit DTH responses in guinea pigs infected with M. tuberculosis or * Corresponding author. W. Yindeeyoungyeon et al. 587M. bovis BCG Tokyo. The results showed that rMPT64 elicits a DTH response comparable to that of PPD in M. bovis BCG Tokyo-vaccinated animals. However, M. tuberculosis-infected animals show less reactivity to rMPT64 than they do to PPD. Although single rCFP10 or rESAT6 did not readily elicit a DTH response in M. tuberculosis-infected animals, combining these antigens with rMPT64 led to an increased DTH response, thus enabling the detection of TB infection.

Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd

“…Purified protein derivative (PPD) from M. bovis (PPDB) and PPD from M. avium (PPDA) (both from IDvet, France) were used as representative MTBC and NTM mixed antigens. Recombinant ESAT6 and CFP-10 were prepared using Escherichia coli and purified by affinity column chromatography as previously described 25 .…”

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus)

“…Recombinant ESAT6 and CFP10 were produced by using Escherichia coli and purified by affinity column chromatography as previously reported [ 19 ]. Recombinant eIFN© was produced in Escherichia coli transformed with an expression plasmid harboring the eIFN© gene obtained from a female Asian elephant.…”