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

Wandee Yindeeyoungyeon; Kamolchanok Rukseree; Siriporn Tungsudjai; Tasanee Panichakul

doi:10.4236/aim.2015.58061

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AiM> Vol.5 No.8, August 2015

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Evaluation of Recombinant Mycobacterium tuberculosis Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs ()

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DOI: 10.4236/aim.2015.58061 1,878 Downloads 2,510 Views Citations

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Wandee Yindeeyoungyeon^1*, Kamolchanok Rukseree², Siriporn Tungsudjai³, Tasanee Panichakul⁴

Affiliation(s)

¹National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.
²Mahidol University, Amnatcharoen Campus, Amnatcharoen, Thailand.
³Faculty of Veterinary Science, Mahidol University, Bangkok, Thailand.
⁴Faculty of Science and Technology, Suan Dusit University, Bangkok, Thailand.

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 M. 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.

KEYWORDS

M. tuberculosis, M. bovis BCG Tokyo, Delayed-Type Hypersensitivity, DTH, MPT64, CFP10, ESAT6

Cite this paper

Yindeeyoungyeon, W. , Rukseree, K. , Tungsudjai, S. and Panichakul, T. (2015) Evaluation of Recombinant Mycobacterium tuberculosis Antigens MPT64, CFP10, and ESAT6 for Delayed-Type Hypersensitivity Responses in Guinea Pigs. Advances in Microbiology, 5, 586-598. doi: 10.4236/aim.2015.58061.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Landi, S. and Held, H.R. (1965) Physicochemical and Biological Studies on Various Preparations of Tuberculin Purified Protein Derivative. Applied Microbiology, 13, 132-139.

[2]	Laal, S. and Skeiky, Y.A.W. (2005) Immune-Based Methods. In: Coke, S.T., Eisenach, K.D., McMurray, D.N. and Jacobs Jr., W.R., Eds., Tuberculosis and the Tubercle Bacillus, ASM Press, Washington DC, 71-83. http://dx.doi.org/10.1128/9781555817657.ch5

[3]	Coler, R.N., Skeiky, Y.A.W., Ovendale, P.J., Vedvick, T.S., Gervassi, L., Guderian, J., Jen, S., Reed, S.G. and Campos-Neto, A. (2000) Cloning of a Mycobacterium tuberculosis Gene Encoding a Purified Protein Derivative Protein That Elicits Strong Tuberculosis-Specific Delayed-Type Hypersensitivity. Journal of Infectious Diseases, 182, 224-233. http://dx.doi.org/10.1086/315677

[4]	Kalra, M., Khuller, G.K., Sheikh, J.A. and Verma, I. (2010) Evaluation of Mycobacterium Tuberculosis Specific RD Antigens for Delayed Type Hypersensitivity Responses in Guinea Pig. Indian Journal of Experimental Biology, 48, 117-123.

[5]	Stavri, H., Bucurenci, N., Ulea, I., Costache, A., Popa, L. and Popa, M.I. (2012) Use of Recombinant Purified Protein Derivative (PPD) Antigens as Specific Skin Test for Tuberculosis. Indian Journal of Medical Research, 136, 799-807.

[6]	Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S. and Small, P.M. (1999) Comparative Genomics of BCG Vaccines by Whole-Genome DNA Microarray. Science, 284, 1520-1523. http://dx.doi.org/10.1126/science.284.5419.1520

[7]	Haga, S., Yamaguchi, R., Nagai, S., Matsuo, K., Yamazaki, A. and Nakamura, R.M. (1995) Delayed-Type Hypersensitivity to a Recombinant Mycobacterial Antigen, MPB64, in Guinea Pigs Sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG. Journal of Leukocyte Biology, 57, 221-225.

[8]	Geisbrecht, B.V., Nikonenko, B., Samala, R., Nakamura, R., Nacy, C.A. and Sacksteder, K.A. (2006) Design and Optimization of a Recombinant System for Large-Scale Production of the MPT64 Antigen from Mycobacterium tuberculosis. Protein Expression and Purification, 46, 64-72. http://dx.doi.org/10.1016/j.pep.2005.08.011

[9]	Woodcock, D.M., Crowther, P.J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S.S., Michael, M.Z. and Graham, M.W. (1989) Quantitative Evaluation of Escherichia coli Host Strains for Tolerance to Cytosine Methylation in Plasmid and Phage Recombinants. Nucleic Acids Research, 17, 3469-3478. http://dx.doi.org/10.1093/nar/17.9.3469

[10]	Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Bubendorff, J.W. (1990) Use of T7 RNA Polymerase to Direct Expression of Cloned Genes. Methods in Enzymology, 185, 60-89. http://dx.doi.org/10.1016/0076-6879(90)85008-C

[11]	Fischer, A.H., Jacobson, K.A., Rose, J. and Zeller, R. (2008) Hematoxylin and Eosin Staining of Tissue and Cell Sections. CSH Protocols.

[12]	Oettinger, T., Holm, A., Mtoni, I.M., Andersen, A.B. and Haslov, K. (1995) Mapping of the Delayed-Type Hypersensitivity-Inducing Epitope of Secreted Protein MPT64 from Mycobacterium tuberculosis. Infection and Immunity, 63, 4613-4618.

[13]	Elhay, M.J., Oettinger, T. and Andersen, P. (1998) Delayed-Type Hypersensitivity Responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the Guinea Pig. Infection and Immunity, 66, 3454-3456.

[14]	Colangeli, R., Spencer, J.S., Bifani, P., Williams, A., Lyashchenko Keen, M.A., Hill, P.J., Belisle, J. and Gennaro, M.L. (2000) MTSA-10, the Product of the Rv3874 Gene of Mycobacterium tuberculosis, Elicits Tuberculosis-Specific, Delayed-Type Hypersensitivity in Guinea Pigs. Infection and Immunity, 68, 990-993. http://dx.doi.org/10.1128/IAI.68.2.990-993.2000

[15]	Lyashchenko, K., Manca, C., Colangeli, R., Heijbel, A., Williams, A. and Gennaro, M.L. (1998) Use of Mycobacterium tuberculosis Complex-Specific Antigens for a Skin Test Specific for Tuberculosis. Infection and Immunity, 66, 3606-3610.

[16]	Wilcke, J.T., Jensen, B.N., Ravn, P., Andersen, A.B. and Haslov, K. (1996) Clinical Evaluation of MPT-64 and MPT-59, Two Proteins Secreted from Mycobacterium tuberculosis, for Skin Test Reagents. Tubercle and Lung Disease, 77, 250-256. http://dx.doi.org/10.1016/S0962-8479(96)90009-X

[17]	Hanif, S.N., Al-Attiyah, R. and Mustafa, A.S. (2010) Species-Specific Antigenic Mycobacterium tuberculosis Proteins Tested by Delayed-Type Hypersensitivity Response. International Journal of Tuberculosis and Lung Disease, 14, 489-494.