A Monoclonal Antibody with a High Affinity for Ricin Isoforms D and E Provides Strong Protection against Ricin Poisoning

Abstract

Ricin is a highly potent toxin that has been used in various attempts at bioterrorism worldwide. Although a vaccine for preventing ricin poisoning (RiVax™) is in clinical development, there are currently no commercially available prophylaxis or treatments for ricin intoxication. Numerous studies have highlighted the potential of passive immunotherapy using anti-ricin monoclonal antibodies (mAbs) and have shown promising results in preclinical models. In this article, we describe the neutralizing and protective efficacy of a new generation of high-affinity anti-ricin mAbs, which bind and neutralize very efficiently both ricin isoforms D and E in vitro through cytotoxicity cell assays. In vivo, protection assay revealed that one of these mAbs (RicE5) conferred over 90% survival in a murine model challenged intranasally with a 5 LD₅₀ of ricin and treated by intravenous administration of the mAbs 6 h post-intoxication. Notably, a 35% survival rate was observed even when treatment was administered 24 h post-exposure. Moreover, all surviving mice exhibited long-term immunity to high ricin doses. These findings offer promising results for the clinical development of a therapeutic candidate against ricin intoxication and may also pave the way for novel vaccination strategies against ricin or other toxins.

Keywords: long-term immunity; monoclonal antibodies; neutralizing antibodies; passive immunotherapy; ricin; ricin isoforms.

Figure 1

Neutralizing capacity of new generation mAbs on Jurkat cells. Ricin was applied at a final concentration sufficient to induce over 95% of cell death, and mAbs at concentrations varying from 500 pg/mL to 100 µg/mL. (a) Neutralization of an equimolar mixture of ricin D and E. Selected anti-ricin mAbs identified in the screening on ricin D + E were evaluated for (b) neutralization of ricin D and (c) neutralization of ricin E. (d) 50% inhibitory concentration (IC₅₀) of mAb measured for each test on Jurkat cells. Each point is a mean ± SD of two independent experiments (a) (except for RicE1, RicE2, RicE3, and 43RCA-G1 (one experiment)) or of technical replicates from one experiment (b,c). ND: not detectable, NT: not tested.

Figure 2

Evaluation of mAb binding specificity to RTA or RTB using sandwich ELISA (a) The binding capacities of RicE4, RicE5, RicE7, RicE8, RA35 (anti-RTA antibody control) and RB34 (anti-RTB antibody control) [27,28] to RTA were assessed. The signal is normalized to the maximum binding measured with RA35 for RTA. (b) The binding capacities of the same mAbs to RTB were evaluated. The signal is normalized to the maximum binding measured with RB34 for RTB.

Figure 3

Neutralization curves of mAbs or mAb combinations of an equimolar mixture of ricin D and E on Jurkat cells. Cells were incubated with the ricin mixture at a final concentration of 10 CD₅₀ and mAbs at concentrations varying from 500 pg/mL to 100 µg/mL for each experiment. Each point is a mean ± SD of at least three independent experiments. (a) Comparison of neutralizing abilities between first-generation and second-generation mAbs. 43RCA-G1, RB34, RicE4, RicE5, RicE8. (b) Neutralizing capacities of RicE5 and RicE8 compared to their equimolar combination RicE5 + RicE8. (c) Neutralizing capacities of RicE5 and RB34 compared to their equimolar combination RicE5 + RB34.

Figure 4

Neutralizing efficacies of RicE5 and RicE8 against ricin D + E in Vero, A549, and Jurkat cells. Each experiment was repeated at least three times. Representative curves of in vitro neutralization of an equimolar ricin D and E solution at 10 CD₅₀ by RicE5 (a) and RicE8 (c). Each point is a mean ± SD. Mean IC₅₀ values ± SEM and mAb:ricin molar ratio at the IC₅₀ ± SEM of RicE5 (b) and RicE8 (d). Statistical analysis: One-way ANOVA with Tukey’s post-hoc test. RicE5 or RicE8 IC₅₀ values or molar ratios for Jurkat cells, are compared to IC₅₀ values or molar ratios for A549 and Vero cells. ns: not statistically significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5

Survival rates of mice treated 6 h post-intoxication. RicE5 and RicE5 + RicE8 were tested in four independent experiments, RicE8 and RB34 + 43RCA-G1 in two independent experiments, and ricin controls in six independent experiments. The other groups were evaluated in a single experiment. The survival rates of the different treatment groups were compared to those of the control group. n = number of mice tested in total (8 to 10 mice per group per experiment). Statistical analysis: Log-rank (Mantel-Cox). ns: not statistically significant, *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

Figure 6

Survival curves of mice treated with RicE5 or RicE5 + RicE8 at 10 mg/kg at various time points (6, 10, 18, and 24 h) following intoxication with 5 LD₅₀ of ricin D + E. The data are pooled from multiple experiments (8 to 10 mice per treatment group per experiment); n = total number of mice treated per group. For the control group exposed to 5 LD₅₀ of ricin, the number of mice per experiment was 5 or 10 mice. Survival rates of the treatment groups were compared with the control group, and comparisons were also made between the different treatments at each time point. Statistical analysis: Log-rank (Mantel-Cox). ns: not statistically significant, ***: p < 0.001; ****: p < 0.0001. The time of injection post-intoxication is indicated in the legend.

Figure 7

Evaluation of circulating anti-ricin antibodies in mouse plasma. (a) Concentration evolution of anti-ricin antibodies in mouse plasma over 300 days. Average concentration (mean ± SEM) of circulating anti-ricin antibodies in the plasma of mice exposed to 5 LD₅₀ of ricin treated with 10 mg/kg, 6 h after intoxication, and which subsequently survived (treatments detailed in the legend, n represents the number of surviving mice per group). Blood samples were collected monthly for 10 months. Statistical analysis: two-way ANOVA was performed using Tukey’s multiple comparison test. Only the statistics for the comparison of the group treated with one mAb (RicE5, orange line) and the group treated with three mAbs (RicE5 + RB34 + 43RCA-G1, blue line) are represented here. Other comparisons are represented in Figure S4. ns: not statistically significant, *: p < 0.05, **: p < 0.01. (b) Concentration evolution of the RicE5 + RicE8 mixture in mice plasma over 300 days (only quantifiable samples are shown). Plasma samples from 10 individual mice were measured using ELISA. Mice received a single i.v. injection of mAbs (RicE5 + RicE8, 10 mg/kg), and blood samples were collected monthly for 10 months. (c) Evaluation of the neutralizing efficacy of an equimolar mixture of ricin D and E at 10 CD₅₀ using pooled plasma. The plasma samples evaluated were collected before the second exposure to ricin. Each point is a mean ± SD of at least two technical replicates from one experiment.

Figure 8

Comparison of clinical signs in mice following initial and second exposure to 5 LD₅₀ ricin 10 months apart. Each point of the graph represents the average clinical score of the surviving mice within each group, plotted as the mean ± SD. For the initial exposure, mice received a dose of 5 LD₅₀ of ricin D + E, followed by treatment with mAbs at 10 mg/kg 6 h later (or PBS for the “ricin control 5 LD₅₀” group). For the second exposure, 10 months after the first exposure, surviving mice were re-exposed to 5 LD₅₀ of ricin via the i.n. route with no subsequent treatment. A “ricin control 5 LD₅₀” group was included for the second exposure as well. No mice from either control group survived. n = number of mice per group that received both exposures. Statistical analysis was performed using a paired t-test to compare the maximum clinical scores across all groups on days 3, 4, and 5 after the initial and the second exposure. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 9

Titration of anti-ricin antibodies in the plasma of mice that survived two exposures to ricin, each at 5 LD₅₀, with a 10-month interval between exposures. Plasma samples from mice that received the same treatment after their initial exposure were pooled for analysis (equal volume from each mouse within the same group; details of the groups are provided in the legend, with n representing the number of mice per group). Blood samples were collected two weeks prior to the second exposure and 21 days after re-exposure. A paired t-test was performed to compare the levels of anti-ricin antibodies before and after the second re-exposure across all groups. **: p < 0.01.

Figure 10

Correlation between the in vivo survival and the IC₅₀ of mAb or mAb combinations against ricin D + E isoform mixture (1:1). The survival rate of mice injected with antibodies at 10 mg/kg 6 h post-ricin exposure was plotted against the mean IC₅₀ value obtained from in vitro neutralization assays in Jurkat cells. Green: protective mAb or mAb combinations, orange: moderately protective, red: not protective.