Date of Award

1-1-2012

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xviii, 221 pages) : illustrations (some color)

Dissertation/Thesis Chair

Nicholas J Mantis

Committee Members

Anne Messer, Christina Egan, Todd A Gray, Charles B Shoemaker

Keywords

Biothreat, Immunology, Ricin, Toxin, Vaccines, Apoptosis, Microbial toxins, Antigenic determinants

Subject Categories

Biology | Immunology of Infectious Disease

Abstract

Ricin toxin's enzymatic subunit (RTA) is a 267 amino acid RNA N-glycosidase that selectively depurinates eukaryotic ribosomal RNA and arrests protein synthesis. The crystal structure of RTA revealed that the protein assumes three distinct folding domains (FD). Residues within FD1 and FD2 form RTA's active site pocket and are proposed to interface with ribosomal proteins, while FD3's primary function is to associate with ricin's B subunit (RTB). In this study I sought to identify the regions of RTA that are important in eliciting toxin-neutralizing antibodies (TNA), as this information is critical for current efforts to develop RTA-based subunit vaccines. I first produced and characterized a large collection of RTA-specific murine monoclonal antibodies (mAbs). I demonstrated that the most potent ricin-neutralizing mAbs recognize at least four distinct epitopes within FD1 and FD2. Two further evaluate the importance of FD1 and FD2 in eliciting protective immunity, I compared an RTA-based subunit vaccine (RVEc) consisting of only FD1 and FD2 to a full length subunit vaccine (RiVax). I found that the two vaccines were equally effective, arguing that FD1 and FD2 are indeed sufficient to elicit complete protection against ricin. The information gained in the first two aims of my dissertation enabled me assist in the design and testing of thermostable variants of RTA, several of which elicited higher total and neutralizing RTA-specific Abs than RiVax when tested in mice. Finally, I provide evidence that RTA-specific mAbs may function intracellularly and that a number of these mAbs, when partially humanized, may have applications for post exposure therapeutics. In summary these findings will significantly advance the development of a vaccine and immuno-therapeutics for ricin and will have important implications for the design of other subunit vaccines for biothreat agents.

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