176 R. Waters and M. Vordermeier
12.4 Application of Specific
Antigens for use in TST and IGRAs
for Diagnostic Purposes and as
a DIVA StrategyExtensive studies have been carried out over
the past 20 years to discover and develop spe-
cific antigens for use in TSTs and IGRAs, as well
as a means to differentiate infected from vacci-
nated animals (DIVA) (reviewed by Schiller
et al., 2010; Vordermeier et al., 2011; Bezos
et al., 2014). Specific antigens may be used in
place of PPDs or in addition to PPDs (i.e.
side-by-side tests), particularly with IGRAs
( Andersen et al., 2000). While numerous anti-
gens have been evaluated, ESAT-6 and CFP10
are currently considered the most immuno-
dominant antigens both for CMI-based tests
and as DIVA reagents with BCG-based vaccines
(Anderson et al., 2000; Pollock et al., 2001;
Vordermeier et al., 2001). Pollock and Ander-
sen were the first to demonstrate the diagnostic
potential of recombinant ESAT-6 in IGRAs in
cattle (Pollock and Andersen, 1997a) and
improved accuracy using recombinant ESAT-6
and CFP10 in combination (van Pinxteren
et al., 2000). The same group then showed the
potential, with limitations, for use of ESAT-6
protein as a skin-test reagent in cattle (Pollock
et al., 2003). Later, it was demonstrated that
ESAT-6 along with MPB64 and MPB83 elicits
IFN-γ responses in M. bovis-infected but not
BCG-vaccinated cattle (Vordermeier et al.,
1999, 2000), and, importantly, peptides of
ESAT-6 and CFP10 can be used as antigens in
IGRAs (Vordermeier et al., 2001). Further,
addition of peptides from other M. bovis anti-
gens (e.g. Rv3873, Rv3879c, Rv0288, and
Rv3019c [Cockle et al., 2006] or Rv3615c
[Sidders et al., 2008; Casal et al., 2012])
improved the sensitivity of ESAT-6/CFP10-
based IGRAs. One caveat to using an ESAT-6/
CFP10 approach, however, is the potential,
albeit uncommon, for cross-reactivity of
ESAT-6 and CFP10 peptides with Mycobacte-
rium kansasii homologues, thereby confound-
ing interpretation of the test in animals
infected with or sensitized to M. kansasii
( Vordermeier et al., 2007). With that said,
infection/sensitization with M. kansasii may
confound interpretation of PPD-based tests as
well (Waters et al., 2006a).
The elucidation of the genome of a number
of mycobacterial species including M. tuberculo-
sis, M. bovis, BCG and M. avium subsp. paratuber-
culosis over the last two decades has allowed a
more rational approach to mine for antigens
recognized by bovine T cells (see section 12.5 for
more details on approaches). However, the
approaches taken were not unbiased, as a num-
ber of hypotheses were tested. For example, anti-
gen prioritization strategies involved listing
potential antigens due to their expression levels,
whether they were predicted to be secreted or
were likely to be induced by hypoxia. These
approaches have been recently reviewed in a
detailed manuscript (Vordermeier et al., 2016).
The leading candidates coming out of these
antigen-mining activities at present for antigens
for diagnostic and DIVA applications are ESAT-6/
CFP10/Rv3615c +/- Rv3020c for use in both
TST and IGRAs (Sidders et al., 2008; Whelan
et al., 2010a; Vordermeier et al., 2011; Jones
et al., 2012). Antigen preparations may include
recombinant proteins or overlapping peptides. A
potential concern with this approach is the rela-
tively high cost of antigen production, particu-
larly for use as a skin-test reagent requiring ~30
mg/dose; however, applying economy of scale
will reduce costs considerably to a degree com-
parable to that of tuberculin. Chen et al. (2014)
and Parlane et al. (2015) have recently devel-
oped a low cost and high production method to
produce polyester beads displaying ESAT-6/
CFP10/Rv3615c +/- Rv3020c proteins on the
surface in a spherical design that theoretically
enhances antigen uptake and presentation by
antigen presenting cells. These antigens could be
used in TST, IGRA or other CMI-based tests. The
high output method of production for these
beads displaying antigen is of particular benefit
for regulatory release of large batches of prod-
uct. Initial trials using ESAT-6/CFP10/Rv3615c
+/- Rv3020c as recombinant proteins, peptide
cocktails or as displayed on polyester beads are
encouraging and further studies are underway
in several countries (e.g. New Zealand, UK and
USA) to better determine the accuracy and prac-
ticality of this approach in the field with both
IGRA and TST assays.