Immunological Diagnosis 177
12.5 Antigen-Mining Strategies for
Discovery of Additional Specific
Antigens of Diagnostic Use12.5.1 Mapping diagnostic antigensOne of the main tasks of TB diagnostic test
development is the identification (mining) of
strong and specific antigens recognized by T cells
from tuberculous animals (infected mainly, but
not exclusively, with M. bovis) but not from
individuals sensitized by environmental myco-
bacterial species, M. avium subsp. paratuberculo-
sis or BCG (if vaccination is being considered). A
number of empirical approaches have been
applied that led to the discovery of the antigens
introduced in section 12.4 (e.g. ESAT-6, CFP-10,
Rv3615c, Rv3020c and others) and we will
discuss these approaches in this section.
12.5.2 Hypothesis-driven approaches
to antigen miningThe elucidation of the genomes for relevant
mycobacterial species (including M. bovis
[ Garnier et al., 2003], M. tuberculosis [Cole et al.,
1998], M. bovis BCG [Brosch et al., 2007],
M. avium subsp. avium and M. avium subsp. para-
tuberculosis [Li et al., 2005]) and the advent
of microarray technology has revolutionized
antigen- mining strategies. The following para-
graphs summarize the most widely applied of
these approaches.
Comparative genomic analysisComparative genomic analysis has been used to
identify M. bovis genes that are deleted from the
genome of BCG (either as individual gene dele-
tions or present in deleted gene regions, the so-
called RD regions), or that contain mutations
resulting in either truncations or modified
amino acid sequences after frame-shifting.
( Pollock and Andersen 1997a, 1997b; Ravn
et al., 1999; van Pinxteren et al., 2000;
(^) Vordermeier et al., 2001). Thus, antigens such
as ESAT-6 and CFP-10 have the capacity to
differentiate M. bovis-infected cattle from
BCG-vaccinated cattle (Buddle et al., 1999;
Vordermeier et al., 1999, 2001). The DIVA
potential of other gene products encoded in the
RD1 region and other regions (RD2 and RD14)
deleted from the BCG genome (Garnier et al.,
2003; Brosch et al., 2007) has also been assessed
(Cockle et al., 2002, 2006) but none of the anti-
gens identified in these studies complemented
ESAT-6 and CFP-10 in increasing overall test
sensitivity in cattle. Thus, alternative approaches
to comparative genomic analysis were needed to
identify potential DIVA antigens to complement
ESAT-6/CFP-10 and increase overall test
sensitivity.
Comparative transcriptome analysis
This approach has been used to explore the link
between gene-expression levels and antigenicity.
M. tuberculosis and M. bovis gene products that
were consistently expressed at high levels under
a variety of culture conditions (known as the
abundant invariome) (Sidders et al., 2007) were
tested in cattle. These studies identified one anti-
gen, Rv3615c, which was recognized by a
significant proportion of infected animals but
not in BCG vaccinates (Sidders et al., 2008). Fur-
thermore, Rv3615c responses were detected in a
proportion of cattle not detected by ESAT-6/
CFP-10 (i.e. Rv3615c complemented ESAT-6/
CFP-10 to increase overall test sensitivity;
Sidders et al., 2008).
Bacterial cell biology
In TB research, it has long been held that secre-
tion of antigenic proteins by mycobacteria
induces strong cellular immune responses in the
host. To identify potential DIVA reagents, 119
M. bovis proteins predicted to be secreted were
screened in infected cattle and BCG vaccinates
(Jones et al., 2010b, 2010c). These studies con-
firmed the immune dominance of members of
the ESAT-6 protein family (Jones et al., 2010b).
The ESAT-6 family member Rv3020c, associated
with the esx-3 secretion site, showed DIVA
potential in cattle (Jones et al., 2010c). However,
subsequent evaluation of these proteins in a
larger cohort of infected animals failed to
demonstrate complementation of the ESAT-6/