Published Ahead of Print 12 December 2012.
2013, 20(2):181. DOI:
Clin. Vaccine Immunol.
J. Brennan and John S. Spencer
Hee Jin Kim, Kalyani Prithiviraj, Nathan Groathouse, Patrick
Quantitative Real-Time PCR
Mycobacterium leprae by Using
Hypothetical Unknown Proteins of
Immunological Evaluation of Unique
Gene Expression Profile and
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Gene Expression Profile and Immunological Evaluation of Unique
Hypothetical Unknown Proteins of Mycobacterium leprae by Using
Quantitative Real-Time PCR
Hee Jin Kim, Kalyani Prithiviraj, Nathan Groathouse, Patrick J. Brennan, John S. Spencer
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific an-
tigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diag-
markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins
six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6
(ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were
expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients
and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune
lesions (1; http://www.who.int/lep/diagnosis/en/index.html), but
due to low sensitivity, physical diagnosis is applicable only to pa-
tients with actual disease. More than 70% of infected patients are
negative for acid-fast bacilli (AFB) and do not present analgesic
skin lesions, especially paucibacillary/tuberculoid (PB/TT) lep-
rosy patients (1). Since the presence of skin lesions in these pa-
cifically diagnose leprosy (1). These problems are accentuated in
the case of diagnosis of individuals with subclinical Mycobacte-
rium leprae infection, including household contacts (HHCs) of
leprosy patients, regarded as the primary source of ongoing lep-
rosy prevalence (1–5).
Several M. leprae antigens have been identified and evaluated
for their diagnostic potential by serological or cell-mediated im-
M. leprae-specific antigen, phenolic glycolipid I (PGL-I), success-
the majority of PB/TT patients and HHCs, though these individ-
uals present strong CMI responses to mycobacterial antigens (3,
13). Both in vitro gamma interferon release assays (IGRAs) and a
simple delayed-type hypersensitivity skin test have been devel-
oped to detect individuals in the early stages of leprosy, using
highly antigenic M. leprae fractions and the major individual im-
application of IGRAs to the major M. leprae protein antigens is
that most of these antigens share appreciable homology with or-
thologues in Mycobacterium spp. in general, resulting in undesir-
able cross-reactivity in individuals such as those vaccinated with
M. bovis BCG or exposed to M. tuberculosis or nontuberculous
toms, requiring the presence of a neurological deficit and skin
Comparative genomic analyses of M. leprae and other myco-
bacteria have identified up to 142 hypothetical unknown open
teins or synthetic peptides originating from these ORFs and con-
taining T cell epitopes restricted via major HLA-DR alleles have
been studied as M. leprae-specific antigens (6, 7, 14–17, 22), and
some have been shown by IGRAs to differentiate individuals in-
(16, 17). However, the levels of gamma interferon (IFN-?) secre-
often too low to distinguish all individuals exposed to M. leprae
sensitivity of current IGRAs raises the question of whether any of
the hypothetical unknowns are expressed or are immunologically
relevant, especially considering that about 50% of M. leprae genes
are pseudogenes (26; http://genolist.pasteur.fr/Leproma/; http:
the host immune system, to eventually be used as diagnostic re-
Received 3 July 2012 Returned for modification 25 July 2012
Accepted 29 November 2012
Published ahead of print 12 December 2012
Address correspondence to Hee Jin Kim, Hee.Kim@colostate.edu.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
February 2013 Volume 20 Number 2Clinical and Vaccine Immunologyp. 181–190 cvi.asm.org
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