4A,Fig. 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. == Introduction == The gastrointestinal (GI) tract is the site of numerous microbial infections caused by a range of pathogens, including:Helicobacter pylori,SalmonellaTyphi,Vibrio cholerae,Escherichia coli,Campylobacter jejuni, andC. difficile. The current approach for treating most of these infections involves administration of antibiotics, which places selection pressure on the organism, can lead to antibiotic resistance, and suppresses or eliminates beneficial commensal microbes. Disease-causing pathogens of the GI tract rely on Fmoc-Val-Cit-PAB-PNP a myriad of virulence factors for colonization, adherence, motility, cellular entry, and pathogenesis. These include, but are not limited to: surface-layer proteins, adhesins, invasins, flagella, high-molecular weight toxins, and quorum sensing molecules. Inhibition of bacterial virulence factors that are essential for disease pathogenesis therefore represents a novel, nonantibiotic based strategy to treat infectious diseases, while reducing the risk of microbial resistance and maintaining commensal gut populations[1],[2],[3]. Several approaches are Fmoc-Val-Cit-PAB-PNP being explored for antivirulence microbial therapy. Inhibition ofE. colipilus assembly[4],Bacillus anthracislethal factor[5],[6], Type III secretion systems[7],[8],Staphylococcus aureusquorum sensing pathways[9], cholera toxin[10]andC. difficiletoxins A and B[11],[12], with small molecules and peptides, are examples currently under development. One of the most pursued antivirulence strategies is targeting bacterial toxins with antibodies. Neutralizing antibodies against anthrax[13], shiga toxin[14], cholera toxin[15], botulinum toxin[16]andC. difficiletoxins[17],[18],[19],[20],[21]have all been successfully isolated and a number of clinical trials involving antibodies to bacterial targets are underway[22]. OCLN For human pathogens that secrete toxins into the GI lumen before cellular entry, such asC. difficile[23], it may be advantageous to neutralize the toxins within the GI tract. Several studies indicate that oral administration of immunoglobulins (i.e., bovine Ig, human IgA, chicken IgY) may be successful at controlling various GI pathogens, includingC. difficile[21],[24], rotavirus[25], shigella[26], and enterotoxigenicE. coliin humans[27]and Fmoc-Val-Cit-PAB-PNP neonatal pigs[28]. However, there are major limitations facing orally administered immunotherapeutics, including the susceptibility of antibodies to proteolytic degradation, instability at low pH, high dosing requirements and cost[29]. Recombinant antibody fragments, such as single-domain antibodies (sdAbs)[30],[31]isolated from conventional IgGs (i.e., VHs, VLs), from the heavy-chain IgG ofCamelidaespecies (i.e., VHHs) and from cartilagous shark IgNARs (i.e., VNARs), are ideal agents to explore for oral immunotherapy[32]because of their small size (12 kDa15 kDa), high affinity, high protease and thermal stability, high expression, amenability to library selection under denaturing conditions for isolating superstable species and ease of genetic manipulation. Despite possessing relatively high intrinsic protease and pH stability, a limited number of studies have shown that, when administered orally, sdAbs are readily degraded in the low pH pepsin-rich environment of the stomach and by digestive enzymes in the duodenum[33],[34],[35]. Several.