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Camelid-derived, single-domain antibodies (V H Hs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed V H H library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique V H Hs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent V H Hs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The V H Hs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines., Competing Interests: The authors declare no conflict of interest.

During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (V H Hs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such V H Hs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these V H Hs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these V H Hs rendered cells resistant to ricin intoxication. One V H H (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Typhoid toxin is secreted by the typhoid fever-causing bacterial pathogen Salmonella enterica serovar Typhi and has tropism for immune cells and brain endothelial cells. Here, we generated a camelid single-domain antibody (VHH) library from typhoid toxoid-immunized alpacas and identified 41 VHHs selected on the glycan receptor-binding PltB and nuclease CdtB. VHHs exhibiting potent in vitro neutralizing activities from each sequence-based family were epitope binned via competition enzyme-linked immunosorbent assays (ELISAs), leading to 6 distinct VHHs, 2 anti-PltBs (T2E7 and T2G9), and 4 anti-CdtB VHHs (T4C4, T4C12, T4E5, and T4E8), whose in vivo neutralizing activities and associated toxin-neutralizing mechanisms were investigated. We found that T2E7, T2G9, and T4E5 effectively neutralized typhoid toxin in vivo , as demonstrated by 100% survival of mice administered a lethal dose of typhoid toxin and with little to no typhoid toxin-mediated upper motor function defect. Cumulatively, these results highlight the potential of the compact antibodies to neutralize typhoid toxin by targeting the glycan-binding and/or nuclease subunits.

Camelid-derived and synthetic single-domain antibodies (sdAbs) are emerging as potent weapons against the novel coronavirus, SARS-CoV-2. sdAbs are small, compact, thermostable immunoglobulin elements capable of binding targets with subnanomolar affinities. By leveraging the power of phage- and yeast surface-display technologies, rare sdAbs can be isolated from highly diverse and complex antibody libraries. Once in hand, sdAbs can be engineered to improve binding affinity, avidity, target specificities, and biodistribution. In this Opinion piece we highlight a series of sophisticated studies describing the identification of ultrapotent sdAbs directed against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. We discuss the possible applications of these antibodies in the global fight against COVID-19., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near-perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (V H Hs) previously reported to recognize epitopes in proximity to RTA's active site. Three of the V H Hs, V2A11, V8E6, and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies ("intrabodies"). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTA's active site and interact with key catalytic residues. V2G10, by contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four V H Hs also penetrated/occluded RTA's active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.toxin, antibody, structure, intracellular., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 Å resolution) revealed extensive antibody contact with RTA's β-strand h (732 Å2 buried surface area; BSA), along with limited engagement with α-helix D (90 Å2) and α-helix C (138 Å2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with α-helix B (82 Å2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin.

We previously produced a heavy-chain-only antibody (Ab) VH domain (V H H)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538-36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific V H Hs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the V H H-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique V H Hs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 V H Hs grouped into more than 20 different competition bins. The RTA-specific V H Hs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific V H Hs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development., (Copyright © 2017 American Society for Microbiology.)

In this report we investigated, within a group of closely related single domain camelid antibodies (V H Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like V H Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1 R29G mutant was largely devoid of toxin-neutralizing activity (TNA). However, the TNA of the V1C7 G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function., (© 2017 Wiley Periodicals, Inc.)

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH "heterodimers." As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.