1. Field of the Invention
The present invention generally relates to ricin toxin. In particular, the present invention relates to ricin vaccines, compositions and therapeutics as well as methods of making and using thereof.
2. Description of the Related Art
Ricin is a very toxic protein obtained from the castor bean, Ricinus communis, Euphorbiaceae. Ricin is a heterodimer comprising an A chain and a B chain joined by a disulfide bond. Ricin A chain (RTA) is an N-glycosidase enzyme that irreversibly damages a specific adenine base from 28S rRNA. Once the rRNA has been damaged, the cell cannot make protein and will inevitably die (cytotoxicity). As RTA exhibits this type of destructive catalytic activity, RTA is commonly referred to as a type II ribosome inactivating protein (RIP). See Lord, et al. (1991) Semin. Cell Biol. 2(1):15–22. RTA has been coupled with a targeting moiety to selectively destroy target cells such as tumor cells. See U.S. Pat. Nos. 4,980,457; 4,962,188; and 4,689,401; see also Vitetta et al. (1993) Trends Pharmacol. Sci. 14:148–154 and Ghetie & Vitetta (1994) Cancer Drug Delivery 2:191–198.
The toxic consequences of ricin are due to the biological activity of RTA. Ricin B chain (RTB) binds the toxin to cell surface receptors and then RTA is transferred inside the cell where inhibition of ribosome activity occurs. The human lethal dose of ricin toxin is about 1 μg/kg. As highly purified ricin is readily available using methods known in the art, the use of ricin toxin in biological warfare and terrorism is highly possible and probable.
Ricin toxin (RT) or Ricin communis agglutinin II (RCA 60), a glycoprotein produced by the castor bean plant, Ricin communis, is composed of two subunits, about a 30 kDa enzymatically active A subunit (RTA) and about a 32 kDa B subunit (RTB). See Lord & Roberts (1994) Faseb J. 8(2):201–208. The B-chain mediates receptor binding of the toxin to eukaryotic cells via its high affinity for galactose. See Alami & Taupiac (1997) Cell Biol. Int. 21(3): 145–150. Once internalized within the cell, the A chain causes catalytic depurination of the 28S ribosomal RNA that results in inhibition of protein synthesis. See Chen & Link (1998) Biochemistry 37(33):11605–11613. Ricin is highly toxic and can cause death when given in sufficient quantities by either systemic or inhalational routes of exposure. See Wilhelmsen & Pitt (1996) Vet. Pathol. 33(3):296–302.
Ricin is a Category B Agent on the Centers for Disease Control (CDC) Select Agent List and thus there is a strong interest in developing diagnostic tests for toxin identification in clinical and environmental samples. See Thomas, M. (2002) “Possession, use, and transfer of select agents and toxins; interim final rule.” Federal Register 240(67). In addition, because there is no vaccine for ricin and no therapeutic agents available for treatment, there is a serious need to develop prophylactic and therapeutic countermeasures for ricin intoxication.
Development of antibodies recognizing determinants on the ricin molecule may be able to address several of these needs. Not only can antibodies be used for diagnostic reagents, but they can also neutralize the toxin by either preventing binding to cells or inhibiting enzymatic activity. There is evidence to suggest that antibodies can protect against ricin intoxication as animals were protected from lethality by administration of polyclonal antibody prior to exposure to ricin. See Hewetson & Rivera (1993) Vaccine 11(7):743–746; and Houston (1982) J. Clin. Toxicol. 19(4):385–9. Anti-ricin IgG has also been shown to protect against inhalational challenge in animals, demonstrating the feasibility of using antibody to protect against this route of exposure as well. See Griffiths & Lindsay (1995) Hum. Exp. Toxicol. 14(2):155–164; and Poli & Rivera (1996) Toxicon. 34(9):1037–1044.
Although polyclonal antibody can be used for these purposes, monoclonal antibodies offer several potential advantages, including consistency and reproducibility of product and the ability to humanize the antibody molecule to reduce adverse reactions caused such as serum sickness when animal antibodies are used therapeutically. Several monoclonal antibodies (Mab) previously developed have been shown to confer protection against ricin intoxication in vitro. See Colombatti & Johnson (1987) J. Immunol. 138(10):3339–33344; Colombatti & Pezzini (1986) Hybridoma 5(1):9–19; and Columbatti (1997) Personal communication to M. Dertzbaugh. However these Mabs were lost several years ago and to date only one Mab that still exists which has been shown to protect against ricin intoxication in vivo and this Mab is directed towards the A chain of the holotoxin. See Lemley & Amanatides (1994) Hybridoma 13(5):417–421.
Thus, a need exists for Mabs against ricin toxin.