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Comparative Study
.2021 Jan 29;13(2):100.
doi: 10.3390/toxins13020100.

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars

Affiliations
Comparative Study

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars

Maria Lucia Orsini Delgado et al. Toxins (Basel)..

Abstract

Ricin, a highly toxic protein fromRicinus communis, is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.

Keywords: Ricinus communis cultivars; monoclonal antibodies; mouse model; neutralizing antibodies; pulmonary intoxication; recombinant antibodies; ricin; ricin isoforms; treatment.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Determination of in vitro cytotoxicity of different ricin isoforms or ricin purified from different cultivars. Jurkat cells were incubated in the presence of different ricin concentrations. Ricin (0–1 ng/mL) was incubated with 104 cells/mL and cell viability was assessed after 72 h by comparison of ATP levels in each well with mean levels of the ATP in control wells (cells without ricin) using the CellTiter-Glo® Luminescence Cell Viability Assay kit (Promega, Madison, United States). Cytotoxicity curves for ricin D (black circles, solid line), E (grey squares, dashed line), or D+E (1:1) (light-grey triangles, dotted line) fromR. communis cultivar (cv.) carmencita, ricin purified fromR. communis cv. zanzibarensis (blue hexagon, solid line) and fromR. communis cv. sanguineus (red inverted triangle, solid line). Mean curves and standard deviation (SD) of three independent experiments.
Figure 2
Figure 2
Affinity of antibodies (Abs) for ricin isoforms D and E. Anti-mouse Fc (AMC) sensors were loaded with one of the mouse monoclonal Abs (mAbs) RB34 or RB37 and anti-human Fc (AHC) sensors were loaded with the humanized recombinant Ab (rAb) 43RCA-G1. The loaded sensors were then dipped in wells containing serial dilutions of ricin D or E, followed by immersion in a running buffer. Kinetic curves of affinity for (a) RB34/ricin D; (b) RB37/ricin D; (c) 43RCA-G1/ricin D; (d) RB34/ricin E; (e) RB37/ricin E; (f) 43RCA-G1/ricin E.
Figure 3
Figure 3
Effect of the mAbs RB34 and RB37 or lactose as control on the binding of ricin to and internalization in Vero cells. Vero cells were incubated with fluorescent ricin D (ricin FP532, 1 µg/mL) pre-incubated with RB34 or RB37 (500 µg/mL). Lactose and a non-neutralizing anti-RTB mAb (RB22) were used as controls. Binding (incubation for 30 min at 4 °C, panelsa toe) and internalization (incubation for 4 h at 37 °C, panelsf toj) of fluorescent ricin D was evaluated through the incubation of cells with ricin alone (panelsa andf) or in presence of RB34 (panelsb andg), RB37 (panelsc andh), lactose (panelsd andi), or the non-neutralizing anti-RTB mAb RB22 (panelse andj).
Figure 4
Figure 4
Pharmacokinetic study of RB34 and 43RCA-G1 in mice. Purified Ab (50 µg) was injected by the intraperitoneal (i.p.) route into BALB/cJ mice (n = 4 for each time-point). Mice were sacrificed at different times to calculate the plasma concentration of Abs RB34 (green filled circles) and 43RCA-G1 (violet filled triangles) using an immunoassay.
Figure 5
Figure 5
In vivo protective activity of RB34 and 43RCA-G1 Abs alone or combined. Female BALB/cJ mice were instilled with 5 LD50 of ricin D+E by the intranasal (i.n.) route and treated by i.p. administration of 10 mg/kg of RB34 (green dashed-dotted line), 43RCA-G1 (violet dashed line), RB34+43RCA-G1 (pink solid line), or PBS (black dotted line) as the control, (a) concomitantly (b) 6 or (c) 24 h after poisoning. Mean curves of two independent experiments (n = 10 for each experiment). Statistical analysis: log-rank (Mantel-Cox) tests, ns: not significant, *:p < 0.05, **:p < 0.01.
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