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Journal of Clinical Microbiology, September 2006, p. 3086-3093, Vol. 44, No. 9
0095-1137/06/$08.00+0     doi:10.1128/JCM.02672-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Enhanced Patient Serum Immunoreactivity to Recombinant Mycobacterium tuberculosis CFP32 Produced in the Yeast Pichia pastoris Compared to Escherichia coli and Its Potential for Serodiagnosis of Tuberculosis

Chaouki Benabdesselem,¹ Dahmani M. Fathallah,¹ Richard C. Huard,^2, Hongxia Zhu,² Mohamed Ali Jarboui,¹ Koussay Dellagi,¹ John L. Ho,^2* and Ridha M. Barbouche^1*

Laboratory of Immunopathology, Vaccinology, and Molecular Genetics, Institut Pasteur de Tunis, Tunis, Tunisia,¹ Division of International Medicine and Infectious Diseases, Department of Medicine, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York²

Received 23 December 2005/ Returned for modification 13 February 2006/ Accepted 11 June 2006

CFP32 is a Mycobacterium tuberculosis complex-restricted secreted protein that was previously reported to be present in a majority of sputum samples from patients with active tuberculosis (TB) and to stimulate serum antibody production. CFP32 (originally annotated as Rv0577 and also known as TB27.3) was therefore considered a good candidate target antigen for the rapid serodiagnosis of TB. However, the maximal sensitivity of CFP32 serorecognition may have been limited in earlier studies because recombinant CFP32 (rCFP32) produced in Escherichia coli was used as the test antibody-capture antigen, a potential shortcoming stemming from differences in bacterial protein posttranslational modifications. To further investigate the serodiagnostic potential of rCFP32 synthesized in different heterologous hosts, we expressed rCFP32 in the yeast Pichia pastoris. Compared to E. coli rCFP32, yeast rCFP32 showed a higher capacity to capture polyclonal antisera in Western blot studies. Likewise, yeast rCFP32 was significantly better recognized by the sera from TB patients and healthy Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals, by enzyme-linked immunosorbent assay (ELISA), than E. coli rCFP32. In subsequent testing, the yeast rCFP32-based antibody-capture ELISA had a sensitivity of 85% and a specificity of 98% for the discrimination of active TB cases (n = 40) from BCG vaccinees (n = 39). The sensitivity was surprisingly high for a single-antigen TB serodiagnostic test compared to tests using E. coli-expressed antigens. Overall, the trans-production of rCFP32 in P. pastoris significantly improved the serologic detection of CFP32-specific antibodies in patient sera, thereby offering a new, possibly better, modality for producing antigens of diagnostic potential for use in the development of immunoassays for both TB and other infectious diseases.

Present address: Clinical Microbiology Service and the Department of Pathology, Columbia University Medical Center, NewYork-Presbyterian Hospital, New York, New York.

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