A pilot clinical trial of a recombinant ricin vaccine in normal humans

Abstract

Ricin, a highly potent toxin produced by castor beans, is classified by the Centers for Disease Control and Prevention as a level B biothreat because it is easily produced, readily available, and highly stable. There have been >750 cases of documented ricin intoxication in humans. There is no approved vaccine for ricin. Ricin contains a lectin-binding B chain and a ribotoxic A chain (RTA). In addition to its ribotoxic site, we have identified a separate site on RTA that is responsible for inducing vascular leak syndrome (VLS) in humans. We have generated a recombinant RTA with two amino acid substitutions that disrupt its ribotoxic site (Y80A) and its VLS-inducing site (V76M). This mutant recombinant RTA (named RiVax) was expressed and produced in Escherichia coli and purified. When RiVax was injected i.m. into mice it protected them against a ricin challenge of 10 LD50s. Preclinical studies in both mice and rabbits demonstrated that RiVax was safe. Based on these results, we have now conducted a pilot clinical trial in humans under an investigational new drug application submitted to the Food and Drug Administration. In this study, three groups of five normal volunteers were injected three times at monthly intervals with 10, 33, or 100 mug of RiVax. The vaccine was safe and elicited ricin-neutralizing Abs in one of five individuals in the low-dose group, four of five in the intermediate-dose group, and five of five in the high-dose group. These results justify further development of the vaccine.

Fig. 1.

Ab responses of the individual volunteers. The Ab titers of each participant from groups 1 and 2 (A) (volunteer 5, ▴; volunteer 6, ▵; volunteer 7, ■; volunteer 9, ♦; volunteer 10, ◇) and group 3 (B) (volunteer 11, ▴; volunteer 12, ▵; volunteer 13, ■; volunteer 14, □; volunteer 15, ♦). Time 0 is the time of study entry. The vaccinations were given on days 0, 28, and 56 except where noted in Results.

Fig. 2.

Ribbon diagram of Ricin holotoxin and RiVax. (A) Ricin with the A chain (blue) with key active site residues (white), the B chain (green), and interchain and intrachain disulfide bonds (orange). (B) RiVax with the active site residues (white), VLS site (orange), the residues that are mutated to inactivate each site, Y80 and V76 (green), and the immunodominant epitopes, 124–140 and 161–195 (yellow). Structures were obtained from GenBank entries 2AAI and 1RTC, respectively, and were drawn by using cncd 4.1 (www.ncbi.nlm.nih.gov).