Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages.

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-ß-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized VH domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered VHs, bearing all possible pairwise combinations of Cys residues in neighboring ß-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in VH FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-ß-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-ß-sheet: Cys6-Cys119) into a diverse panel of VH, VL, and VHH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human VH repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering.

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VHH, VH, or VL) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology.

Carbohydrate esterase family 16 contains fungal hemicellulose acetyl esterases (HAEs) with varying specificity.

Acetyl esterases are an important component of the enzymatic machinery fungi use to degrade plant biomass and are classified in several Carbohydrate Esterase families of the CAZy classification system. Carbohydrate Esterase family 16 (CE16) is one of the more recently discovered CAZy families, but only a small number of its enzyme members have been characterized so far, revealing activity on xylan-derived oligosaccharides, as well as activity related to galactoglucomannan. The number of CE16 genes differs significantly in the genomes of filamentous fungi. In this study, four CE16 members were identified in the genome of Aspergillus niger NRRL3 and it was shown that they belong to three of the four phylogenetic Clades of CE16. Significant differences in expression profiles of the genes and substrate specificity of the enzymes were revealed, demonstrating the diversity within this family of enzymes. Detailed characterization of one of these four A. niger enzymes (HaeA) demonstrated activity on oligosaccharides obtained from acetylated glucuronoxylan, galactoglucomannan and xyloglucan, thus establishing this enzyme as a general hemicellulose acetyl esterase. Their broad substrate specificity makes these enzymes highly interesting for biotechnological applications in which deacetylation of polysaccharides is required.

Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.

Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction.

The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis.

KEY MESSAGE: A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology. Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC.

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: ∼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.

Isolation and characterization of camelid single-domain antibodies against HER2.

OBJECTIVE: To isolate and characterize novel high-affinity llama single-domain antibodies against human HER2. RESULTS: We immunized a llama with human HER2, constructed a phage-displayed VHH library from the lymphocytes of the animal, and isolated six unique HER2-specific VHHs by panning. All six VHHs were unique at the amino acid level and were clonally unrelated, as reflected by their distinct CDR3 lengths. All six VHHs recognized recombinant human HER2 ectodomain with monovalent affinities ranging from 1 to 51 nM, had comparable affinities for cynomolgus monkey HER2, and bound HER2+ SKOV3 cells by flow cytometry. Three of the VHHs recognized recombinant murine HER2 with no loss of affinity compared with human and cynomolgus monkey HER2. The VHHs recognized three major epitopes on HER2 (including one conserved across the human, simian and murine orthologues), all of which were distinct from that of trastuzumab. These VHHs may be useful in the design of modular cancer immunotherapeutics.

Oxford nanopore sequencing enables rapid discovery of single-domain antibodies from phage display libraries.

Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies' MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Here, we applied nanopore sequencing to single-domain Ab (sdAb) repertoires and phage-displayed sdAb libraries. We show that despite low overall data fidelity, sdAb sequences could be reconstructed above a frequency threshold (∼100 copies); however, distinguishing clonal sdAb variants was not always possible. The data quality was sufficient to enable rapid identification of antigen-specific sdAb sequences enriched during panning of phage display libraries, obviating the need for screening single clones.

A disulfide-stabilized human VL single-domain antibody library is a source of soluble and highly thermostable binders.

We have previously shown that incorporation of a second intradomain disulfide linkage into camelid VHH and human VH/VL single-domain antibodies confers increased thermostability. Here, we explored the effects of introducing an additional disulfide linkage, formed between Cys48 and Cys64 (Kabat numbering), into a phage-displayed synthetic human VL library. In comparison to an identical library bearing only the highly conserved Cys23-Cys88 disulfide linkage, the disulfide-stabilized VL library tolerated a similar degree of randomization but retained a higher level of functional diversity after selection with protein L. Both libraries yielded soluble, antigen-specific VLs that recognized a model antigen (maltose-binding protein) with similar affinities, in the micromolar range; however, the disulfide-stabilized antigen-specific VLs were much more thermostable (average ΔTm ∼10°C) than non-disulfide-stabilized VLs. This work provides proof-of-concept for building synthetic antibody libraries using disulfide-constrained immunoglobulin domains, thus avoiding pitfalls of post-hoc disulfide linkage engineering such as impaired antigen binding and reduced expression yield.

Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries.

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.

A Highly Effective Component Vaccine against Nontyphoidal Salmonella enterica Infections.

UNLABELLED: Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections. IMPORTANCE: Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa.

Functional and structural diversity in GH62 α-L-arabinofuranosidases from the thermophilic fungus Scytalidium thermophilum.

The genome of the thermophilic fungus Scytalidium thermophilum (strain CBS 625.91) harbours a wide range of genes involved in carbohydrate degradation, including three genes, abf62A, abf62B and abf62C, predicted to encode glycoside hydrolase family 62 (GH62) enzymes. Transcriptome analysis showed that only abf62A and abf62C are actively expressed during growth on diverse substrates including straws from barley, alfalfa, triticale and canola. The abf62A and abf62C genes were expressed in Escherichia coli and the resulting recombinant proteins were characterized. Calcium-free crystal structures of Abf62C in apo and xylotriose bound forms were determined to 1.23 and 1.48 Å resolution respectively. Site-directed mutagenesis confirmed Asp55, Asp171 and Glu230 as catalytic triad residues, and revealed the critical role of non-catalytic residues Asp194, Trp229 and Tyr338 in positioning the scissile α-L-arabinofuranoside bond at the catalytic site. Further, the +2R substrate-binding site residues Tyr168 and Asn339, as well as the +2NR residue Tyr226, are involved in accommodating long-chain xylan polymers. Overall, our structural and functional analysis highlights characteristic differences between Abf62A and Abf62C, which represent divergent subgroups in the GH62 family.

Quantitative mass spectrometry catalogues Salmonella pathogenicity island-2 effectors and identifies their cognate host binding partners.

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

Salmonella phage ST64B encodes a member of the SseK/NleB effector family.

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.

Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes.

Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 A resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

Pathogenic adaptation of intracellular bacteria by rewiring a cis-regulatory input function.

The acquisition of DNA by horizontal gene transfer enables bacteria to adapt to previously unexploited ecological niches. Although horizontal gene transfer and mutation of protein-coding sequences are well-recognized forms of pathogen evolution, the evolutionary significance of cis-regulatory mutations in creating phenotypic diversity through altered transcriptional outputs is not known. We show the significance of regulatory mutation for pathogen evolution by mapping and then rewiring a cis-regulatory module controlling a gene required for murine typhoid. Acquisition of a binding site for the Salmonella pathogenicity island-2 regulator, SsrB, enabled the srfN gene, ancestral to the Salmonella genus, to play a role in pathoadaptation of S. typhimurium to a host animal. We identified the evolved cis-regulatory module and quantified the fitness gain that this regulatory output accrues for the bacterium using competitive infections of host animals. Our findings highlight a mechanism of pathogen evolution involving regulatory mutation that is selected because of the fitness advantage the new regulatory output provides the incipient clones.

SseL is a salmonella-specific translocated effector integrated into the SsrB-controlled salmonella pathogenicity island 2 type III secretion system.

Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.

Negative regulation of Salmonella pathogenicity island 2 is required for contextual control of virulence during typhoid.

Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic pathogenesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection. ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early "gain-of-virulence" dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo.