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. Author manuscript; available in PMC: 2014 Sep 1.
Published in final edited form as: Toxicon. 2013 Apr 17;72:29–34. doi: 10.1016/j.toxicon.2013.04.005

Neutralizing Activity and Protective Immunity to Ricin Toxin Conferred by B Subunit (RTB)-Specific Fab Fragments

Anastasiya Yermakova 1,2, Nicholas J Mantis 1,2,*
PMCID: PMC3774546  NIHMSID: NIHMS502816  PMID: 23603317

Abstract

SylH3 and 24B11 are murine monoclonal antibodies directed against different epitopes on ricin toxin’s binding (RTB) subunit that have been shown to passively protect mice against ricin challenge. Here we report that Fab fragments of SylH3 and 24B11 neutralize ricin in a cell based assay, and in a mouse challenge model as effectively as their respective full length parental IgGs. These data demonstrate that immunity to ricin can occur independent of Fc-mediated clearance.

Keywords: Toxin, Neutralizing Antibodies, Biodefense, Mouse

Ricin is a member of the A–B family of protein toxins, which includes cholera toxin, Shiga toxin (Stx), botulinum neurotoxins (BoNT) and anthrax toxin. Ricin’s B subunit (RTB) is a galactose- and N-acetylgalactosamine (Gal/GalNac) lectin that promotes toxin attachment and entry into virtually all mammalian cell types (Rutenber et al., 1987; Sandvig et al., 1976). RTB also mediates the intracellular retrograde trafficking of ricin from the plasma membrane to the endoplasmic reticulum (ER). Ricin’s A subunit (RTA) is an RNA N-glycosidase (RTA) whose sole substrate is a universally conserved adenosine residue within the so-called sarcin/ricin loop (SRL) of mammalian ribosomal RNA (Spooner and Lord, 2012). Hydrolysis of the SRL by RTA results in the cessation of cellular protein synthesis, activation of the ribotoxic stress response (RSR), and cell death via apoptosis (Jandhyala et al., 2012).

Structurally, RTB consists of two globular domains with identical folding topologies (Montfort et al., 1987). Each of the two domains (1 and 2) is comprised of three homologous sub-domains (α, β, γ) that probably arose by gene duplication from a primordial carbohydrate recognition domain (CRD) (Montfort et al., 1987; Rutenber et al., 1987). However, only the external sub-domains, 1α and 2γ, retain functional carbohydrate recognition activity (Rutenber et al., 1987; Swimmer et al., 1992). Subdomain 1α (residues 17–59) is Gal-specific and is considered a “low affinity” CRD, whereas sub-domain 2γ (residues 228–262) binds both Gal and GalNac and is considered a “high affinity” CRD (Newton et al., 1992; Rutenber and Robertus, 1991; Zentz et al., 1978). Either sub-domain 1α or 2γ, which are separated by approximately 70 Angstroms, are sufficient to promote toxin attachment to cells (Montfort et al., 1987).

Our laboratory has recently produced and characterized a large collection of RTB-specific murine monoclonal antibodies (mAbs) (McGuinness and Mantis, 2006; Yermakova and Mantis, 2011; Yermakova et al., 2012). The majority of these mAbs fail to neutralize ricin, even though they bind ricin holotoxin with high affinities. For example, TFTB-1 has an affinity of >5 × 10−9 M, yet has no demonstrable capacity to inactivate ricin in vitro or in vivo (Yermakova and Mantis, 2011). To date we have identified only three RTB-specific mAbs (24B11, SylH3 and JB4) that are capable of neutralizing ricin in vitro and able to passively protect mice against a lethal toxin challenge. All three are IgG1s and each bind ricin with nanomolar affinities (SylH3, 3.38×10−9 M; 24B11, 4.2×10−9 M; JB4, 2.01×10−10 M) (Yermakova and Mantis, 2011; Yermakova et al., 2012). 24B11’s epitope has been tentatively localized within RTB’s sub-domain 1α (McGuinness and Mantis, 2006). We speculate that SylH3 and JB4 bind a similar or overlapping epitope in RTB’s sub-domain 2γ (Yermakova et al., 2012).

We consider SylH3 and JB4 as being class I antibodies as they are very effective at blocking ricin binding to cell surfaces, suggesting they work by steric hindrance (Yermakova and Mantis, 2011). We consider 24B11 a class II antibody, as it, neutralizes ricin in cell-based assays as effectively as SylH3 and JB4 but only partially affects toxin attachment to cell surfaces or surrogate receptors like asialofetuin (ASF). We therefore postulate that 24B11 neutralizes ricin at a step downstream of attachment.

We wished to investigate the role of the fragment crystallizable (Fc) components of RTB-specific class I and class II Abs. In vitro, the Fc components of class I Abs, by virtue of their ability to obstruct one or both of RTB CRDs, may be important in steric hindrance and interference with toxin attachment to cell surfaces (McGuinness and Mantis, 2006). In vivo, the Fc-determinants of both class I and class II Abs could assist in toxin clearance via Fcγ receptor (FcγR)-dependent mechanisms. In the case of anthrax toxin and BoNT, there is evidence that FcγR-mediated clearance is critical in Ab-mediated toxin immunity (Mukherjee et al., 2012; Sepulveda et al., 2010; Verma et al., 2009; Vitale et al., 2006).

To investigate the role of Fc-determinants in ricin neutralization in vitro and in vivo, we prepared Fab fragments of SylH3 (class I) and 24B11 (class II). As a control, we also produced Fab fragments of the non-neutralizing mAb TFTB-1. Fab fragments of SylH3, 24B11 and TFTB-1 were produced using a commercially available ficin-based, mouse IgG1 Fab preparation kit (ThermoScientific, Rockford, IL). SDS-PAGE and SureBlue (ThermoScientific) straining confirmed that each mAb was digested to completion (Fig. S1). Furthermore, the purified Fabs each retained their specificity for ricin and RTB, although reactivity of 24B11 Fab with RTB (but not ricin holotoxin) was slightly diminished, as compared to the full length IgG (Fig. S2).

To determine whether the Fc domains of class I Abs are involved in interfering with ricin from accessing cellular receptors, we compared full length SylH3 with its respective Fab in a ricin-ASF attachment assay. For comparison, 24B11 and TFTB-1 full length IgGs and Fabs were subjected to the same assay. Nunc Maxisorb F96 microtiter ELISA plates (ThermoFisher Scientific) were coated with ASF (0.4 μg/well; EY Laboratories, San Mateo, CA) in PBS for 18 h at 4°C, and then probed with pre-incubated mixtures of biotinylated ricin (Vector Laboratories, Burlingame, CA) and mAbs or Fabs at a range of concentrations (Fig. 1).

Fig. 1. Inhibition of ricin-receptor interactions by SylH3 and 24B11 Fabs.

Fig. 1

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Biotin labeled ricin (770 pM) was mixed with SylH3 (A), 24B11 (B) or TFTB-1 (A, B) mAbs and their respective Fab fragments at the indicated concentrations (x-axis) and then applied to 96-well microtiter plates coated with ASF (83 nM). Biotin-ricin binding to ASF (y-axis) was detected by incubation with avidin-HRP (Sigma-Aldrich Co.) and TMB substrate (KPL, Gaithersburg, MD). Percent ricin binding was defined as OD450 values of each treatment/OD450 of no Ab control × 100. Each symbol (with SEM) represents the average of at least two replicate wells.

As expected, full length SylH3 IgG inhibited, in a dose-dependent manner, the attachment of ricin to ASF. Fifty percent inhibition (IC50) was achieved with ~1.0 nM SylH3, while >90% inhibition (IC90) was achieved with ~15 nM SylH3 (Fig. 1A). Although SylH3 Fab fragments also inhibited ricin attachment to ASF at high concentrations, they were 2–4 fold less effective at lower concentrations than the full length SylH3 IgG. These data suggest that the Fc component of SylH3 is in fact important in inhibiting ricin attachment to ASF. By comparison, 24B11 and 24B11 Fab fragments only partially interfered with ricin attachment to ASF, while TFTB-1 and TFTB-1 Fab fragments had no affect on toxin binding (Fig. 1B).

We next employed a Vero cell (ATCC, Manassas, VA) cytotoxicity assay in order to determine to what degree SylH3 and 24B11 Fab fragments retained the capacity to neutralize ricin in vitro. SylH3 neutralized ricin in a dose-dependent manner, with an IC50 of ~ 15 nM SylH3 (Fig. 2A). With an IC50 of 30 nM, SylH3 Fab fragments were ~2-fold less effective at neutralizing ricin than the respective full length mAb, again arguing for a role of the Fc domain in toxin neutralization by SylH3. In contrast, Fab fragments of 24B11 were equally effective as full-length 24B11 at toxin neutralization (Fig. 2B), thereby confirming that the Fc domain of 24B11 is not required for toxin neutralizing activity in vitro. Finally, neither TFTB-1 nor TFTB-1 Fab fragments had any demonstrable neutralizing activity, as expected (Fig. 2C).

Fig. 2. Neutralizing activity of SylH3, 24B11 and TFTB-1 Fabs.

Fig. 2

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Fab fragments of SylH3 (A), 24B11 (B) and TFTB-1 (C) were assessed for their capacity to protect Vero cells from the cytotoxic effects of ricin and compared to their respective full length IgGs. Vero cells (5×104 cells/ml) were seeded (0.1 ml per well) into white-bottomed 96-well tissue culture plates (Corning Life Sciences, Corning, NY) and allowed to adhere overnight. Cells were then treated with a mixture of 154 pM ricin and varying dilutions of mAb or Fab (starting at 66 nM) for 2 h (x-axis). The cells were then washed and incubated for 48 h before being analyzed for cell viability (y-axis) using CellTiter-GLO (Promega, Madison, WI). Each symbol (with SEM) represents the average of at least two replicate wells.

Having confirmed that SylH3 and 24B11 Fab fragments retain some or all of their ricin-neutralizing activity in vitro, we next sought to examine their ability to passively protect mice against a 10xLD50 toxin challenge. To control for the fact that Fab fragments generally have a shorter half-life in humans and animal models (<20 h) than full-length IgG (~9–30 days) (Bazin-Redureau et al., 1997; Jones et al., 2006; Kim et al., 2008; Ujhelyi and Robert, 1995), mAbs and Fabs were co-administered with ricin toxin (10xLD50) at the time of challenge. Furthermore, to ensure that the molar ratio of toxin to the antibody variable domain (Fv regions) was the same, mice received 60 μg full-length mAbs and 40 μg of Fab fragments. Following challenge, mice were monitored for the onset of hypoglycemia, a well-established surrogate marker of ricin intoxication, as well as mean time to death (Pincus et al., 2002).

The ricin-only group of mice succumbed to intoxication within 24 h, as did the animals treated with ricin and TFTB-1 or TFTB-1 Fab fragments (Table 1; Fig. 3A; Fig. S3). In contrast, mice treated with ricin and concomitantly with SylH3 or SylH3 Fab fragments survived toxin challenge and did not experience a significant drop in blood glucose levels, thereby demonstrating that, at the concentrations tested, both the full-length and Fab fragments were equally effective at neutralizing ricin in vivo. Examination of 24B11-treated mice revealed that 24B11 conferred only partial protection against a 10xLD50 ricin challenge, as evidenced by the fact that one mouse in the 24B11-treated succumbed to ricin intoxication while the surviving mice experienced a significant reduction in blood glucose levels at 24 hr post challenge (Table 1; Fig. 3B,C; Fig. S3). The Fc-domain of 24B11 is apparently not involved in neutralizing ricin in vivo, as 24B11 Fab fragments were as effective as the full length IgG at protecting mice against 10xLD50 ricin challenge. These data demonstrate that the capacities of SylH3 and 24B11 to neutralize ricin in vivo are both Fc-independent.

Table 1.

Onset and recovery from ricin intoxication following challenge with SylH3 and 24B11 IgG and Fabs.

Timea SylH3 24B11 TFTB-1 Control
IgG Fab IgG Fab IgG Fab
0 127b ±7 131 ±6 118 ±4 130 ±9 119 ±1 132 ±2 110 ±2
24 76 ±2 81 ±1 60 ±4 67 ±8 x x n.d.
48 92 ±1 106 ±6 61 ±14 70 ±25 x x n.d.
72 99 ±1 103 ±5 70 ±23 86 ±7 x x n.d.
96 127 ±7 123 ±6 102 ±2 106 ±4. x x n.d.
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a

Time is hours post ricin challenge.

b

Average blood glucose (mg/dl) of mice per group, per time point with standard errors. X, indicates that there were no survivors at these time points.

Fig. 3. Passive protection conferred by SylH3 and 24B11 Fab fragments.

Fig. 3

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Groups of 10 week old female BALB/c mice (Taconic Labs, Germantown, NY) were passively immunized with Fabs (4 μM or 40 μg Fab/animal) or full IgG mAbs (2 μM or 60 μg mAb/animal) by intraperitoneal injection and then challenged 24 h later with 10xLD50s of ricin. (Panel A) Kaplan-Meier survival plot with SyH3, 2B11 and TFTB-1 mAbs and Fab fragments. (Panels B, C) Hypoglycemia was used as a surrogate marker of ricin intoxication, as described previously (Yermakova and Mantis, 2011). Blood glucose levels were measured at just prior to or 24 h after ricin challenge: (Panel B) SylH3, 24B11, TFTB-1 IgGs, (Panel C) SylH3, 24B11, TFTB-1 Fabs, (Panel D) ricin challenge control. Each group contained n = 4 mice. Both 24B11 and SylH3 were tested more than four times in passive protection studies. The Fab fragments were tested for neutralizing activity in a series of in vitro and in vivo pilot studies with 2–3 mice per group and were then compared directly to their respective full length IgGs in the study shown in this figure. P values were obtained using a two-tailed paired t test with a 95% confidence interval.

The fact that Fab fragments of SylH3 and 24B11 were capable of protecting mice against a lethal dose toxin challenge demonstrates that ricin neutralization, at least by these two RTB-specific mAbs, is Fc-independent. While we cannot exclude the possibility that antibody (Ab) constant regions may influence the half-life or tissue distribution of toxin-immune complexes, our data are in accordance with other studies suggesting that ricin neutralization is primarily dictated by Fv-specificity (Vance and Mantis, 2012; Yermakova and Mantis, 2011; Yermakova et al., 2012). For example, non-neutralizing, high-affinity mAbs against RTA or RTB (e.g., TFTB-1) afford no protection against toxin challenge in a mouse model. Nor do oligoclonal mixtures of non-neutralizing mAbs provide any degree of protection (A. Yermakova and N. Mantis, unpublished results). This is in contrast to what has been observed in the case of BoNT where Fc receptor-mediated clearance is important in counteracting high-dose toxin exposure (Nowakowski et al., 2002; Sepulveda et al., 2010) and in the case of anthrax toxin where protection is modulated by IgG subclass and FcγR utilization (Abboud et al., 2010; Harvill et al., 2008; Mabry et al., 2005; Maynard et al., 2002; Wild et al., 2003).

One limitation of this study is that we did not examine RTA-specific mAbs in parallel. Nonetheless, such experiments would be highly informative, as more than a dozen RTA-specific toxin neutralizing mAbs have been described (O’Hara et al., 2010; O’Hara et al., 2012b). A number of these RTA-specific mAbs have been shown to be highly effective at protecting mice when administered prior to, concomitantly, or even as much as 6 h after ricin challenge (O’Hara et al., 2010; O’Hara et al., 2012a; Roche et al., 2008), In general, mAbs directed against RTA have little affect on ricin’s ability to associate with host cell receptors, suggesting that like 24B11 they may neutralize ricin at a step downstream of attachment (Maddaloni et al., 2004; Neal et al., 2010; O’Hara et al., 2010).

In conclusion, the demonstration that Fab fragments of single specificity are sufficient to neutralize ricin in vivo raises the possibility that single chain Abs like camelid Nanobodies (VHHs) may have therapeutic potential. While single chain antibodies have much shorter half-lives that full length human or chimerized mAbs, they do have the advantage of greater tissue penetration and longer shelf-lives (Sepulveda et al., 2010). Thus, future studies will be aimed at evaluating the use of RTB-specific Fabs or single chain Abs as post exposure therapeutics for ricin.

Supplementary Material

01

Fig. S1. SDS-PAGE analysis of digested SylH3, 24B11, and TFTB-1 Fabs under reducing conditions. Each sample was adjusted to 2 μg protein/20 μl (9 μl sample, 9 μl Laemli buffer, 2 μl 2M 2-Mercaptoethanol (BME). Samples were boiled for 10 minutes prior to loading on a 10% SDS Gel. Gels were run in 1x SDS electrophoresis buffer for 30 m at 55 mA, rinsed with water and stained with Gel Code Blue for 30 m 2x, then de-stained overnight; SylH3 (A), TFTB-1 (B), and 24B11 (C). Lane 1 – Precision Plus Protein™ Kaleidoscope standard (Bio-rad, Hercules, CA), lane 2 – reduced Fab (heavy and light chain), lane 3 – reduced IgG (heavy and light chain).

Fig. S2. Reactivity profiles of individual mAbs or Fabs with RTB and ricin holotoxin. Ninety-six well microtiter plates were coated with RTB (left panel), or ricin holotoxin (right panel) and then probed with mAbs (A) SylH3, (B) 24B11, or (C) TFTB-1 or their respective Fab fragments at indicated concentrations (66 nM). 24B11 and TFTB-1 served as RTB-specific controls. Fab or IgG reactivity with RTB or ricin holotoxin measured with OD450. Each symbol (with SEM) represents the average of at least three replicate wells.

Fig. S3. Passive protection conferred by SylH3 and 24B11 Fab fragments or full length IgG as determine by blood glucose levels. Groups of 10 week old female BALB/c mice (Taconic Labs, Germantown, NY) were passively immunized with Fabs (4 μM or 40 μg Fab/animal) or full IgG mAbs (2 μM or 60 μg mAb/animal) by intraperitoneal injection and then challenged 24 h later with 10xLD50s of ricin. Hypoglycemia was used as a surrogate marker of ricin intoxication, as described previously (Yermakova and Mantis, 2011). Blood glucose levels were measured at just prior to or 24 h after ricin challenge: (A) SylH3 vs. SylH3 Fabs, (B) 24B11 vs. 24B11 Fabs, (C) TFTB-1 vs. TFTB-1 Fabs. R (+) denotes ricin challenge control mice that received ricin but no mAb. Each group contained n = 4 mice. The data in this figure are from a single representative experiment.

Acknowledgments

This work was supported by grant AI097688 (PI-Mantis) from the National Institutes of Health. AY was supported in part by a pre-doctoral fellowship from the Wadsworth Center’s Biodefense and Emerging Infectious Diseases program (T32AI055429; PI-McDonough).

Footnotes

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References Cited

  1. Abboud N, Chow SK, Saylor C, Janda A, Ravetch JV, Scharff MD, Casadevall A. A requirement for FcgammaR in antibody-mediated bacterial toxin neutralization. J Exp Med. 2010;207:2395–2405. doi: 10.1084/jem.20100995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bazin-Redureau MI, Renard CB, Scherrmann JM. Pharmacokinetics of heterologous and homologous immunoglobulin G, F(ab’)2 and Fab after intravenous administration in the rat. J Pharm Pharmacol. 1997;49:277–281. doi: 10.1111/j.2042-7158.1997.tb06795.x. [DOI] [PubMed] [Google Scholar]
  3. Harvill ET, Osorio M, Loving CL, Lee GM, Kelly VK, Merkel TJ. Anamnestic protective immunity to Bacillus anthracis is antibody mediated but independent of complement and Fc receptors. Infect Immun. 2008;76:2177–2182. doi: 10.1128/IAI.00647-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Jandhyala DM, Thorpe CM, Magun B. Ricin and Shiga toxins: effects on host cell signal transduction. Curr Top Microbiol Immunol. 2012;357:41–65. doi: 10.1007/82_2011_181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jones RG, Alsop TA, Hull R, Tierney R, Rigsby P, Holley J, Sesardic D. Botulinum type A toxin neutralisation by specific IgG and its fragments: a comparison of mouse systemic toxicity and local flaccid paralysis assays. Toxicon. 2006;48:246–254. doi: 10.1016/j.toxicon.2006.05.007. [DOI] [PubMed] [Google Scholar]
  6. Kim J, Bronson CL, Wani MA, Oberyszyn TM, Mohanty S, Chaudhury C, Hayton WL, Robinson JM, Anderson CL. Beta 2-microglobulin deficient mice catabolize IgG more rapidly than FcRn- alpha-chain deficient mice. Exp Biol Med (Maywood) 2008;233:603–609. doi: 10.3181/0710-RM-270. [DOI] [PubMed] [Google Scholar]
  7. Mabry R, Rani M, Geiger R, Hubbard GB, Carrion R, Jr, Brasky K, Patterson JL, Georgiou G, Iverson BL. Passive protection against anthrax by using a high-affinity antitoxin antibody fragment lacking an Fc region. Infect Immun. 2005;73:8362–8368. doi: 10.1128/IAI.73.12.8362-8368.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Maddaloni M, Cooke C, Wilkinson R, Stout AV, Eng L, Pincus SH. Immunological characteristics associated with the protective efficacy of antibodies to ricin. J Immunol. 2004;172:6221–6228. doi: 10.4049/jimmunol.172.10.6221. [DOI] [PubMed] [Google Scholar]
  9. Maynard JA, Maassen CB, Leppla SH, Brasky K, Patterson JL, Iverson BL, Georgiou G. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat Biotechnol. 2002;20:597–601. doi: 10.1038/nbt0602-597. [DOI] [PubMed] [Google Scholar]
  10. McGuinness CR, Mantis NJ. Characterization of a novel high-affinity monoclonal immunoglobulin G antibody against the ricin B subunit. Infect Immun. 2006;74:3463–3470. doi: 10.1128/IAI.00324-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Montfort W, Villafranca JE, Monzingo AF, Ernst SR, Katzin B, Rutenber E, Xuong NH, Hamlin R, Robertus JD. The three-dimensional structure of ricin at 2.8 A. Journal of Biological Chemistry. 1987;262:5398–5403. [PubMed] [Google Scholar]
  12. Mukherjee J, Tremblay JM, Leysath CE, Ofori K, Baldwin K, Feng X, Bedenice D, Webb RP, Wright PM, Smith LA, Tzipori S, Shoemaker CB. A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS One. 2012;7:e29941. doi: 10.1371/journal.pone.0029941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Neal LM, O’Hara J, Brey RN, 3rd, Mantis NJ. A monoclonal immunoglobulin G antibody directed against an immunodominant linear epitope on the ricin A chain confers systemic and mucosal immunity to ricin. Infect Immun. 2010;78:552–561. doi: 10.1128/IAI.00796-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Newton DL, Wales R, Richardson PT, Walbridge S, Saxena SK, Ackerman EJ, Roberts LM, Lord JM, Youle RJ. Cell surface and intracellular functions for ricin galactose binding. J Biol Chem. 1992;267:11917–11922. [PubMed] [Google Scholar]
  15. Nowakowski A, Wang C, Powers DB, Amersdorfer P, Smith TJ, Montgomery VA, Sheridan R, Blake R, Smith LA, Marks JD. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody. Proc Natl Acad Sci U S A. 2002;99:11346–11350. doi: 10.1073/pnas.172229899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. O’Hara JM, Neal LM, McCarthy EA, Kasten-Jolly JA, Brey RN, 3rd, Mantis NJ. Folding domains within the ricin toxin A subunit as targets of protective antibodies. Vaccine. 2010;28:7035–7046. doi: 10.1016/j.vaccine.2010.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O’Hara JM, Whaley K, Pauly M, Zeitlin L, Mantis NJ. Plant-based expression of a partially humanized neutralizing monoclonal IgG directed against an immunodominant epitope on the ricin toxin A subunit. Vaccine. 2012a;30:1239–1243. doi: 10.1016/j.vaccine.2011.12.058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. O’Hara JM, Yermakova A, Mantis NJ. Immunity to ricin: fundamental insights into toxin-antibody interactions. Curr Top Microbiol Immunol. 2012b;357:209–241. doi: 10.1007/82_2011_193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pincus SH, Eng L, Cooke CL, Maddaloni M. Identification of hypoglycemia in mice as a surrogate marker of ricin toxicosis. Comp Med. 2002;52:530–533. [PubMed] [Google Scholar]
  20. Roche JK, Stone MK, Gross LK, Lindner M, Seaner R, Pincus SH, Obrig TG. Post-exposure targeting of specific epitopes on ricin toxin abrogates toxin-induced hypoglycemia, hepatic injury, and lethality in a mouse model. Lab Invest. 2008;88:1178–1191. doi: 10.1038/labinvest.2008.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rutenber E, Ready M, Robertus JD. Structure and evolution of ricin B chain. Nature. 1987;326:624–626. doi: 10.1038/326624a0. [DOI] [PubMed] [Google Scholar]
  22. Rutenber E, Robertus JD. Structure of ricin B-chain at 2.5 A resolution. Proteins. 1991;10:260–269. doi: 10.1002/prot.340100310. [DOI] [PubMed] [Google Scholar]
  23. Sandvig K, Olsnes S, Pihl A. Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells. J Biol Chem. 1976;251:3977–3984. [PubMed] [Google Scholar]
  24. Sepulveda J, Mukherjee J, Tzipori S, Simpson LL, Shoemaker CB. Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents. Infect Immun. 2010;78:756–763. doi: 10.1128/IAI.01084-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Spooner RA, Lord JM. How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum. Curr Top Microbiol Immunol. 2012;357:19–40. doi: 10.1007/82_2011_154. [DOI] [PubMed] [Google Scholar]
  26. Swimmer C, Lehar SM, McCafferty J, Chiswell DJ, Blattler WA, Guild BC. Phage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants. Proc Natl Acad Sci U S A. 1992;89:3756–3760. doi: 10.1073/pnas.89.9.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ujhelyi MR, Robert S. Pharmacokinetic aspects of digoxin-specific Fab therapy in the management of digitalis toxicity. Clin Pharmacokinet. 1995;28:483–493. doi: 10.2165/00003088-199528060-00006. [DOI] [PubMed] [Google Scholar]
  28. Vance DJ, Mantis NJ. Resolution of two overlapping neutralizing B cell epitopes within a solvent exposed, immunodominant alpha-helix in ricin toxin’s enzymatic subunit. Toxicon. 2012;60:874–877. doi: 10.1016/j.toxicon.2012.06.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Verma A, Ngundi MM, Meade BD, De Pascalis R, Elkins KL, Burns DL. Analysis of the Fc gamma receptor-dependent component of neutralization measured by anthrax toxin neutralization assays. Clin Vaccine Immunol. 2009;16:1405–1412. doi: 10.1128/CVI.00194-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vitale L, Blanset D, Lowy I, O’Neill T, Goldstein J, Little SF, Andrews GP, Dorough G, Taylor RK, Keler T. Prophylaxis and therapy of inhalational anthrax by a novel monoclonal antibody to protective antigen that mimics vaccine-induced immunity. Infect Immun. 2006;74:5840–5847. doi: 10.1128/IAI.00712-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wild MA, Xin H, Maruyama T, Nolan MJ, Calveley PM, Malone JD, Wallace MR, Bowdish KS. Human antibodies from immunized donors are protective against anthrax toxin in vivo. Nat Biotechnol. 2003;21:1305–1306. doi: 10.1038/nbt891. [DOI] [PubMed] [Google Scholar]
  32. Yermakova A, Mantis NJ. Protective immunity to ricin toxin conferred by antibodies against the toxin’s binding subunit (RTB) Vaccine. 2011;29:7925–7935. doi: 10.1016/j.vaccine.2011.08.075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yermakova A, Vance DJ, Mantis NJ. Sub-Domains of Ricin’s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies. PLoS One. 2012;7:e44317. doi: 10.1371/journal.pone.0044317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zentz C, Frenoy JP, Bourrillon R. Binding of galactose and lactose to ricin. Equilibrium studies. Biochim Biophys Acta. 1978;536:18–26. doi: 10.1016/0005-2795(78)90047-8. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

01

Fig. S1. SDS-PAGE analysis of digested SylH3, 24B11, and TFTB-1 Fabs under reducing conditions. Each sample was adjusted to 2 μg protein/20 μl (9 μl sample, 9 μl Laemli buffer, 2 μl 2M 2-Mercaptoethanol (BME). Samples were boiled for 10 minutes prior to loading on a 10% SDS Gel. Gels were run in 1x SDS electrophoresis buffer for 30 m at 55 mA, rinsed with water and stained with Gel Code Blue for 30 m 2x, then de-stained overnight; SylH3 (A), TFTB-1 (B), and 24B11 (C). Lane 1 – Precision Plus Protein™ Kaleidoscope standard (Bio-rad, Hercules, CA), lane 2 – reduced Fab (heavy and light chain), lane 3 – reduced IgG (heavy and light chain).

Fig. S2. Reactivity profiles of individual mAbs or Fabs with RTB and ricin holotoxin. Ninety-six well microtiter plates were coated with RTB (left panel), or ricin holotoxin (right panel) and then probed with mAbs (A) SylH3, (B) 24B11, or (C) TFTB-1 or their respective Fab fragments at indicated concentrations (66 nM). 24B11 and TFTB-1 served as RTB-specific controls. Fab or IgG reactivity with RTB or ricin holotoxin measured with OD450. Each symbol (with SEM) represents the average of at least three replicate wells.

Fig. S3. Passive protection conferred by SylH3 and 24B11 Fab fragments or full length IgG as determine by blood glucose levels. Groups of 10 week old female BALB/c mice (Taconic Labs, Germantown, NY) were passively immunized with Fabs (4 μM or 40 μg Fab/animal) or full IgG mAbs (2 μM or 60 μg mAb/animal) by intraperitoneal injection and then challenged 24 h later with 10xLD50s of ricin. Hypoglycemia was used as a surrogate marker of ricin intoxication, as described previously (Yermakova and Mantis, 2011). Blood glucose levels were measured at just prior to or 24 h after ricin challenge: (A) SylH3 vs. SylH3 Fabs, (B) 24B11 vs. 24B11 Fabs, (C) TFTB-1 vs. TFTB-1 Fabs. R (+) denotes ricin challenge control mice that received ricin but no mAb. Each group contained n = 4 mice. The data in this figure are from a single representative experiment.

NIHMS502816-supplement-01.pdf (9.5MB, pdf)

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