4 | Discussion
In recent years, advances in mAb discovery and production have ushered
in the development of pathogen-specific mAbs to be used either per se as
antibacterial drugs or to be integrated into various diagnostic
platforms for the detection of specific pathogens. In the latter regard,
the high affinity and specificity of mAbs are characteristics that can
be exploited in diagnostic tools giving reduced false positive/negative
results. Such tools could provide rapid and accurate identification of
bacterial agents at points of care, thus supporting better clinical
management of patients and preventing the transmission of infectious
diseases in the community.
With regard to the development of mAbs as antibacterial drugs, we note
that the advantages mAb-based antibacterials derive from three main
factors: their relatively low risk of damaging the human microbiome due
to their exquisite specificity; their extended half-life, which could
offer long-term protection; and – according to mounting preclinical
data – their ability to act synergistically with antibiotics (Domenech,
Sempere, de Miguel, & Yuste, 2018; Felts, Grainger, & Slunt, 2000).
Therefore, mAbs may be considered as prime candidates in the fight
against antibiotic-resistant bacteria. Yet, currently there are only a
handful of antimicrobial mAbs under development, most of which target
specific bacterial virulence factors (Dickey et al., 2017), whereas our
work characterizes an antibody that targets the virulence delivery
system.
Although there are still no clinically approved antibacterial mAbs for
the treatment of antibiotic-resistant bacterial infections, previous
attempts have been made to target various components of the T3SS
complex. For example, a study showing that MEDI3902, a bi-specific
anti-PcrV/Ps1 antibody, offers protection against P. aeruginosain animal infection models has provided proof of concept that targeting
an essential component of the T3SS can inhibit bacterial virulence
(DiGiandomenico et al., 2014). Similarly, an anti-SpuE antibody was
recently demonstrated to inhibit the expression of T3SS and thereby to
attenuate the virulence of P. aeruginosa (Zhang et al., 2019).
Here, we describe a mAb raised against EspB, an essential component
within the T3SS that is crucial for the infectivity of numerous
Gram-negative bacteria, including EPEC. Our results demonstrate that
mAb-EspB-B7 binds EspB with high affinity and specificity. The antibody
binding to EspB was stable over a wide range of pH values, excluding
acidic pH values, and across various salt concentrations. A reduced
binding capacity was detected only under high salt concentrations
(> 400 mM), suggesting that the antibody-antigen binding
interface is governed by electrostatic interactions. This idea is
supported by the observation that the identified EspB epitope contains
nearly 50% of charged amino acids, which might be involved in the
antibody-antigen binding. mAb-EspB-B7 demonstrated a relatively high
melting temperature, which was moderately elevated when the antibody was
complexed with its antigen. This result suggests that EspB binding has a
stabilizing effect on the antibody, as was previously reported for
anti-ricin neutralizing antibody (Legler et al., 2017). Furthermore, the
melting temperature profile of mAb-EspB-B7 showed three distinct events
that probably correspond to the melting order of the CH2
region, followed by the Fab and CH3, as reported
previously (Vermeer & Norde, 2000). This melting profile indicates that
the mAb-EspB-B7 would be suitable for applications that require
relatively high thermal stability.
Epitope mapping using our specially designed cyclic-peptide array
revealed that mAb-EspB-B7 binds mostly to a specific amino acid sequence
located at positions 193-210 along the EspB sequence. In a previous
study, it was shown that this region was not important for EspB-EspD
interactions (Luo & Donnenberg, 2011), a fact that was further
corroborated by our observation that mAb-EspB-B7 does not disrupt the
interaction between the two proteins. Moreover, the observation that
mAb-EspB-B7 binds EspB as a component of the fully assembled T3SS
complex supports the notion that the epitope of EspB is exposed and not
buried within the EspB-EspD interface. It is noteworthy that the peptide
array results also identified an additional region, corresponding to
peptides #9-12, that demonstrated mAb-EspB-B7 binding. This finding
could perhaps suggest that the epitope recognized by mAb-EspB-B7 is
conformational rather than linear. As the main epitope sequence
(positions 193-210) is fully conserved in EPEC and C. rodentium ,
the lower similarity along this second region might provide an
explanation for the reduced western blot signal that we observed forC. rodentium EspB (Figure 6A). In addition, while we observed
mAb-EspB-B7 binding to a protein in the supernatants of WT EHEC andC. rodentium , no binding was detected in the Salmonellasupernatant. This result is in agreement with the presence of the
epitope in EHEC and C. rodentium but not in Salmonella(Figure 6B).
The ability of mAb-EspB-B7 to recognize and bind C. rodentiumEspB is highly important, as it provides the scientific grounds for the
use of a mouse model in future studies examining mAb-EspB-B7 protection
against infection. While mAb-EspB-B7 did not demonstrate a reduction of
bacterial infectivity in the ex vivo system, we posit that
examining it in a mouse model will provide a more comprehensive picture
that will include the effect of the antibody in promoting certain
activities of the immune system against bacteria, such as opsonization
and phagocytic clearance. These activities may prevent the spread of the
bacterial infection within the host body and induce a humoral response
with serological memory that will shorten the infection duration,
promote recovery and provide cellular and serological memory.
Another key aspect of mAb-EspB-B7 is its ability to bind both the
secreted form of EspB and EspB as a component of the assembled T3SS
complex within the bacterial cell. This finding provides further support
for its potential as a diagnostic agent capable of detecting bacterial
infections directly in clinical samples in a short time with high
accuracy, as previously reported (Barreiros dos Santos et al., 2013;
Joung et al., 2013).
In summary, we characterized a mAb, namely, mAb-EspB-B7, that binds with
high affinity and selectivity to a T3SS-exposed protein. Future work
with this antibody should focus on testing it as a promising candidate
for development as an anti-bacterial drug and/or for diagnostic
applications, such as in a portable standalone antibody-based biosensor.