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We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins.

Sevenmutants of the heavy chain CDR3 variable loop of the Salmonella B O-polysaccharide-specific antibody Fab fragment were characterized thermodynamically using titration microcalorimetry. Results reveal that the glycine at position 102 cannot be replaced while the glycine at position 100 and the tyrosine at position 103 can be substituted with only small effects on binding. Substitution of the histidine at position 101 with acidic amino acids resulted only in a small reduction in binding affinity. Further, the calorimetric analysis employed revealed tremendous thermodynamic alterations otherwise masked by the similarity in binding constants of the mutants.

Several carbohydrate antigens were raised against chlamydial lipopolysaccharide in presence of high resolution to determine the structures of antigen-binding fragments from two related monoclonal antibodies. The results for antibody-antigen structures provide insight into the mechanism of immune system that used the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes and the unrelated CDR H3 sequences that could result in convergent binding of clinically relevant bacterial antigens.

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (VHHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing VHHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 VHHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as KD = 70 pM, none of the VHHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with VHH-VHH dimers and to a much greater extent with VHH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several VHH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other VHH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the VHHs described here are effective at toxin B neutralization when formatted as bivalent VHH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site. [ABSTRACT FROM AUTHOR]

Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 μM (average: 2-3 μM), but had highly variable expression yields (range: 0.1-19 mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries. [ABSTRACT FROM AUTHOR]