Use of Multiepitope Polyproteins in Serodiagnosis of Active Tuberculosis

Abstract

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ϳ98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ϳ93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.Tuberculosis (TB) is a major disease in developing countries and an increasing problem in developed countries, with an estimated 8 million new cases each year (7,21,35). The rise in the number of infections is due to drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and to more incidences of coinfections with human immunodeficiency virus (HIV), particularly in sub-Saharan Africa (21). This has prompted the need to develop rapid, inexpensive, but clinically sensitive and specific tests that can improve upon current diagnostic tests used in TB diagnosis, such as culture and acidfast smear testing (20). PCR methods are available but are expensive, require culture, and are not applicable to field use (14, 33-34, 42). One such approach to providing simple tests has been to develop adjunctive serology assays. However, previous attempts to diagnose TB by serology have met with limited success (2-3, 6,8, 10-12, 15-16, 23-24, 37-38, 44-47). Many of the antigens to date do not have the appropriate clinical sensitivity and specificity required for an accurate diagnosis and do not always effectively discriminate Mycobacterium bovis BCG-vaccinated and purified protein derivative (PPD)-positive individuals from those with active TB (8, 36). Several notable antigens have been identified which have merit in TB diagnosis, among which is the immunodominant 38-kDa phosphate transport protein (1,5,9,19). This antigen has been used ...

Interferon Gamma Response to Combinations 38 kDa/CFP‐10, 38 kDa/MPT‐64, ESAT‐6/MPT‐64 and ESAT‐6/CFP‐10, Each Related to a Single Recombinant Protein of Mycobacterium tuberculosis in Individuals from Tuberculosis Endemic Areas

“…1). Similar results were reported with the chimera construction expressing 38 kDa, CFP-10 and MTB-8 (17).…”

Combined use of Western blot/ELISA to improve the serological diagnosis of human tuberculosis

“…The TbF6/DPEP antigen association showed better results, with reagent serum samples in 44 (85%) of the patients. This sensitivity is in agreement with that of other studies for the same association using serum from Brazilian patients [21] and is similar to the sensitivity described by Cole et al [28] in a study carried out on patients with pulmonary tuberculosis in China. These authors found a sensitivity of 89% for smear-positive patients, and of 74% for smear-negative patients for the 38 kDa antigen.…”

“…It is a polyprotein designed to incorporate four genes of interest (Mtb8, 38 kDa, Mtb11 and Mtb 48) into a single expression vector [21]. The recombinant antigen DPEP (Corixa® Co.) is a proline-rich protein that has been identified as the MPT 32 antigen.…”

Crystal structure of calcium dodecin (Rv0379), from Mycobacterium tuberculosis with a unique calcium‐binding site

“…Although the biological function of Ca dodecin is yet to be determined, identification of Ca dodecin as an immunodominant sero-antigen emphasizes a possible role in host response. 24,25 The antigenicity of Ca dodecin and its use in detecting Mtbspecific sera from TB patients 26 also suggests that Ca dodecin could be a secreted or shed antigen. 27,28 In addition, the characteristics of the calcium-binding pocket in Ca dodecin suggest that the dodecins could be used as a scaffold to create custom designed metal and flavin binding sites.…”