Neutralising Antibodies against Ricin Toxin

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention

Point-of-care diagnostics for ricin exposure

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Pharmacokinetic study of RB34, RB37 and RA36 in mice.

<p>Purified antibody (50 µg) was injected intraperitoneally into Swiss mice (n = 4). Mice were sacrificed at different times to calculate plasma concentration of mAbs: RB34 (▪); RB37 (▴) and RA36 (•), using an immunoassay.</p

<i>In vivo</i> neutralising activity of anti-ricin antibodies combination pre-incubated with ricin (A) Survival curve.

<p>CD1 mice were intranasally challenged with 5 LD₅₀ of ricin alone (Δ) or pre-incubated with antibodies: several doses of RA36, RB34 and RB37 mixture (Abs) were assessed to obtain antibody/ricin molar ratios of 2 (▪), 5 (•), 10 (▴) and 20 (♦), compared with a nonspecific antibody (ns Ab) in the same concentration (R = 2 (<b>+</b>), R = 5 (<b>X</b>), R = 10 (○), R = 20 (□)). 50 µl of ricin-antibody complex was administered per mouse and mortality was monitored for 21 days. (<b>B</b>) Weight change. In the same experiment, mice were weighed at 0, 2, 7, 14 and 21 days. The percentage of the D0 weight (100%) was calculated, with female CD1 mice as control (▾). Mice injected with ricin alone (Δ) or with mixtures of specific antibodies at R = 2 (▪); R = 5 (•); R = 10 (▴) and R = 20 (♦). The data are representative of two independent experiments.</p

Inhibition of retrograde transport protects mice from lethal ricin challenge.

International audienceBacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target

Affinity constants of RB34, RB37 and RA36 for ricin.

<p>*K_D was calculated from k_on and k_off with n = 3 for each antibody.</p

<i>In vivo</i> neutralising activity of anti-ricin antibodies combination administered after ricin challenge.

<p>(<b>A</b>) Survival curve. CD1 mice were intranasally challenged with 5 LD₅₀ of ricin alone (Δ) or ricin followed by intravenous injection of 5 mg/kg of antibodies 10 min (▪), 1 h (•), 5 h (♦), 7.5 h (▴), 10 h (○) and 24 h (□) after challenge. (<b>B</b>) Weight change. In the same experiment, mice were weighed at 0, 2, 7, 14 and 21 days, taking the weight at day zero as reference (100%), for female CD1 mice as a control (▾), mice injected with ricin (Δ), or ricin followed by intravenous injection of 5 mg/kg of antibodies 10 min (▪), 1 h (•), 5 h (♦), 7.5 h (▴), 10 h (○) and 24 h (□) after challenge. The data are representative of two independent experiments.</p

Combination neutralising effect of antibodies against ricin <i>in vitro</i>.

<p>(<b>A</b>) Combination of pairs of antibodies, with RB34 as control (RB34: ○; RB34/RB37: ▴; RB34/RA36: ♦; RB37/RA36: ▾; RB27/RB37: ▪). (<b>B</b>) Combination of three or four antibodies, with RB34 as control (RB34: ○; RB27/RB37/RA36: •; RB34/RB37/RA36: ▴; RB34/RB37/RA36/RA32: <b>+</b>; RB34/RB37/RA36/RA33: ▪; RB34/RB37/RA36/RA35: ♦). Antibodies were premixed in equimolar ratio at several concentrations (0–10 µg/ml) and incubated with ricin (0.1 ng/ml) before exposure to Jurkat cells. Cell viability was assessed by means of luminescence assay using a Cell titer Glo luminescence kit (Promega).</p

<i>In vivo</i> evaluation of each neutralising anti-ricin antibody administered after ricin challenge.

<p>(<b>A</b>) Survival curve. CD1 mice were intranasally challenged with 5 LD₅₀ of ricin alone (Δ) or ricin followed by intravenous injection of 5 mg/kg of RB34 antibody (▪), RB37 antibody (•), or RA36 antibody (□) 1 h after ricin challenge. (<b>B</b>) Weight change. In the same experiment, mice were weighed at 0, 2, 7, 14 and 21 days, taking the weight at day zero as reference (100%), for female CD1 mice as a control (▾), mice injected with ricin (Δ), or ricin followed by intravenous injection of 5 mg/kg of mouse weight of RB34 antibody (▪), RB37 antibody (•), or RA36 antibody (□) 1 hour after ricin challenge.</p