Individuals received three monthly i.m. 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 g 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. Ricin is an extremely lethal toxin produced by castor beans (1C3). It contains a ribotoxic A chain (RTA) and a cell-binding B chain (1, 4C10). It is widely available, easy to purify, and highly stable as a liquid or powder (11). The estimated lethal dose of ricin in humans is 1C10 g/kg when delivered as an aerosol or by injection (11). In earlier studies, the lethal dose of Trifluridine ricin administered by ingestion was reported to be much higher, i.e., 1C2 g (approximately a teaspoon of powder) (11, 12). Ricin represents a potential agent for use in biological warfare and is classified by the Centers for Disease Control and Prevention as a level B biothreat (13). There is no approved vaccine for ricin. We have developed a recombinant RTA vaccine (14, 15) in which only two amino acids Trifluridine Trifluridine in the protein have been genetically engineered to inactivate both the well known ribotoxic site (5, 16C18) and the recently identified VLS-inducing site (19). The mutant protein, Y80A/V76M or RiVax, lacks both toxic activities but retains all of the immunodominant epitopes recognized by a panel of mAbs (14). Three doses of 1C10 g each administered i.m. to mice in the absence of adjuvant protected them from a subsequent challenge with 10 LD50s of ricin (14, 15). A formal toxicology study in rabbits revealed no toxicity (14). To determine whether RiVax is also safe and immunogenic in humans, we have carried out a pilot clinical trial in which three groups of five volunteers each were vaccinated. Individuals received three monthly i.m. injections of either 10 g (group 1), 33 g (group 2), or 100 g (group 3) of CT5.1 RiVax without adjuvant. The volunteers were monitored for side effects and for the generation of both anti-RTA Abs and ricin-neutralizing Abs. In this report, we present the results of this trial. Results RiVax. RiVax was produced, vialed, and tested in our good manufacturing practice laboratory. Each Trifluridine production run was assigned a lot number, and each lot was tested for release and, at monthly intervals, for stability. Four different lots were used for the vaccinations. The certificate of analysis showing the release criteria is presented in Table 1. Table 1. Vaccine tests and specifications for lot release steadily after the third vaccination, returning to normal levels by day 7. The volunteer denied excessive exercise and the use of either Trifluridine recreational or prescribed drugs; he was asymptomatic. We consider it unlikely that these abnormal values were related to the vaccinations. Titers of Anti-RTA Ab. An RIA was used to measure Ab titers immediately before entry, before each vaccination, 2 weeks after the third vaccination, and at intervals thereafter. The preentry sera were used as negative controls for each postvaccination sample. Each assay was carried out twice. As shown in Table 4, anti-RTA levels at day 70 varied from 0.97C22.6 g/ml and were not related to the dose of vaccine.