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.