Clostridium difficile is a leading cause of nosocomial infection in North AmericaWith favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these V H Hs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. Clostridium difficile is a Gram-positive spore-forming bacillus which is recognized to be the major cause of nosocomial diarrhea associated with antibiotic therapy (35). The incidence of C. difficile infection has been rapidly increasing in both Europe and North America, and this increase in infections has been associated with a significantly high mortality rate (2, 35). The broad spectrum of diseases caused by C. difficile, which range from antibiotic-associated diarrhea to the potentially lethal, pseudomembranous colitis, has been shown to depend on the level of toxin produced (1), and this production is recognized as a critical determinant of pathogenicity. Following antibiotic therapy when the microbiota of the gastrointestinal tract is disrupted, infection by C. difficile is mediated by spores which germinate in the gut, followed by vegetative cell proliferation and the subsequent secretion of the two major virulence factors, the Rho glucosylating toxins TcdA and TcdB.The toxin-encoding genes (tcdA and tcdB) are localized to a 19.6-kb pathogenicity locus (PaLoc) which includes three other accessory genes, tcdR, tcdE and tcdC (43). TcdR is an alternative sigma factor required for transcription of the two toxin genes; TcdE has been described to be a putative holin-like protein involved in toxin secretion, although this role has recently been a source of debate (17, 32); and TcdC is an anti-sigma factor that negatively regulates tcdR-dependent transcription (28, 29). In addition, four other regulators of toxin synthesis have recently been identified: CepA (3), CodY ...

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.

The colocalization of various hKs seems to be essential for the regulation of serine protease activity in skin and for steady desquamation and skin barrier function. Moreover, the increased number of cells expressing multiple KLK mRNA and hK in PV and AD could be a clue to elucidate their pathogenesis.

Background: TcdA and TcdB are the main virulence factors for Clostridium difficile infections. Results: X-ray crystallography, mass spectrometry, and size exclusion chromatography reveal the molecular basis of antibody recognition. Conclusion: Neutralizing antibodies do not directly block binding to known receptors, suggesting new mechanisms of neutralization. Significance: The molecular details of antibody recognition will assist with the development of novel therapeutics and diagnostics.

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Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high affinity (KD range: 1–40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the VHHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VHH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC50: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.

Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VHH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (KD) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC50 of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.