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 RTAand 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 Hdisplayed 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.KEYWORDS antibody, biodefense, epitope, neutralizing, toxins, vaccines R icin is the prototype of the type II ribosome-inactivating protein (RIP) family of toxins (1). Ricin toxin A subunit (RTA) is an RNA N-glycosidase that catalyzes the hydrolysis of a conserved adenine residue within the sarcin/ricin loop (SRL) of 28S rRNA, resulting in ribosome arrest and apoptosis (2-4). Ricin toxin B subunit (RTB) is a galactose-and N-acetylgalactosamine (Gal/GalNAc)-specific lectin that promotes toxin entry into mammalian cells, including the epithelial cells that line the respiratory tract

BackgroundIncreased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells.Methods3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS).Results3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPβ), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control.ConclusionThese results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.Electronic supplementary materialThe online version of this article (10.1186/s12944-017-0574-7) contains supplementary material, which is available to authorized users.

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

Edited by Peter CresswellRicin toxin is a heterodimer consisting of RTA, a ribosomeinactivating protein, and RTB, a lectin that facilitates receptormediated uptake into mammalian cells. In previous studies, we demonstrated that toxin-neutralizing antibodies target four spatially distinct hot spots on RTA, which we refer to as epitope clusters I-IV. In this report, we identified and characterized three single domain camelid antibodies (V H H) against cluster II. One of these V H Hs, V5E1, ranks as one of the most potent ricinneutralizing antibodies described to date. We solved the X-ray crystal structures of each of the three V H Hs (E1, V1C7, and V5E1) in complex with RTA. V5E1 buries a total of 1,133 Å 2 of surface area on RTA and makes primary contacts with ␣-helix A (residues 18 -32), ␣-helix F (182-194), as well as the F-G loop. V5E1, by virtue of complementarity determining region 3 (CDR3), may also engage with RTB and potentially interfere with the high affinity galactose-recognition element that plays a critical role in toxin attachment to cell surfaces and intracellular trafficking. The two other V H Hs, E1 and V1C7, bind epitopes adjacent to V5E1 but display only weak toxin neutralizing activity, thereby providing structural insights into specific residues within cluster II that may be critical contact points for toxin inactivation.

In this report we describe the X-ray crystal structures of two single domain camelid antibodies (VHH), F5 and F8, each in complex with the ricin toxin's enzymatic subunit (RTA). F5 has potent toxin-neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Å2 in complex with RTA and made contact with three prominent secondary structural elements: α–helix B (residues 98-106), β–strand h (residues 113-117), and the C-terminus of α–helix D (residues 154-156). F8 buried 1103 Å2 in complex with RTA that was centered primarily on β–strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions (CDR) were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β–strand within RTA's centrally located β–sheet. A comparison of the two structures reported here to several previously reported (RTA- VHH) structures identifies putative contact sites on RTA, particularly α–helix B, associated with potent toxin-neutralizing activity. This information has implications for rational design of RTA-based subunit vaccines for biodefense.

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Ricin toxin, a plant-derived, mannosylated glycoprotein, elicits an incapacitating and potentially lethal inflammatory response in the airways following inhalation. Uptake of ricin by alveolar macrophages (AM) and other pulmonary cell types occurs via two parallel pathways: one mediated by ricin's B subunit (RTB), a galactosespecific lectin, and one mediated by the mannose receptor (MR;CD206). Ricin's A subunit (RTA) is a ribosomeinactivating protein that triggers apoptosis in mammalian cells. It was recently reported that a single monoclonal antibody (MAb), PB10, directed against an immunodominant epitope on RTA and administered intravenously, was able to rescue Rhesus macaques from lethal aerosol dose of ricin. In this study, we now demonstrate in mice that the effectiveness PB10 is significantly improved when combined with a second MAb, SylH3, against RTB. Mice treated with PB10 alone survived lethal-dose intranasal ricin challenge, but experienced significant weight loss, moderate pulmonary inflammation (e.g., elevated IL-1 and IL-6 levels, PMN influx), and apoptosis of lung macrophages. In contrast, mice treated with the PB10/SylH3 cocktail were essentially impervious to pulmonary ricin toxin exposure, as evidenced by no weight loss, no change in local IL-1 and IL-6 levels, retention of lung macrophages, and a significant dampening of PMN recruitment into the bronchoalveolar lavage (BAL) fluids. The PB10/SylH3 cocktail only marginally reduced ricin binding to target cells in the BAL, suggesting that the antibody mixture neutralizes ricin by interfering with one or more steps in the RTB-and MR-dependent uptake pathways.

In this report we investigated, within a group of closely related single domain camelid antibodies (VHHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like VHHs (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 (i.e., Gly for Arg at position 29) diminished V5C1’s binding affinity by >10 fold. As expected, the V5C1R29G mutant was largely devoid of toxin-neutralizing activity. However, the toxin-neutralizing activity of the V1C7G29R 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.