Toward the Development of an Improved Ricin Vaccine

Abstract

To date, there is no approved antidote to treat or prevent the toxic effects of ricin exposure. RiVax, a recombinant ricin A chain subunit vaccine antigen, is one such antidote being developed as a prophylaxis. While it has been shown to be protective in numerous animal studies, RiVax is currently limited by its ability to elicit a robust toxin-neutralizing antibody (TNA) response. The underlying hypothesis of this dissertation is that a RiVax-based antigen with improved structural stability would result in an enhanced TNA response due to the preservation of conformationally-sensitive epitopes. To that end, two complimentary and orthogonal computational approaches were employed to design twelve point mutations predicted to stabilize the structure of RiVax. Differential scanning calorimetry across a range of pH values revealed seven of the twelve mutations were more stable than RiVax, two had essentially no effect, and three were destabilizing. Serological analysis of mice immunized with RiVax, one of two stabilized mutants, or one of two destabilized mutants suggested that the stabilized antigens induced a qualitatively better immune response. Eight double point mutants and a triple point mutant were then produced by combining the seven stabilizing mutations in various ways. Circular dichroism and fluorescence thermal unfolding curves showed that all nine derivatives were, to varying degrees, more stable than RiVax. Differential scanning calorimetry (DSC) detected two distinct transitions - one which was rather dramatically affected by the mutations and a second which showed more meager gains in stability more in line with the spectroscopic techniques. The first transition was speculated to arise from changes to the rather unstable C-terminal region of RiVax. Serological analysis of mice immunized with RiVax or one of four multi-site derivatives (selected on the basis of highest Tm,1 from DSC analysis) showed that three of the derivatives elicited a more rapid and statistically superior TNA response relative to RiVax. When the mouse study was repeated with a lower antigen dose, a RiVax derivative containing mutations V81I, C171L, and V204I was clearly superior to RiVax and the other three derivatives at eliciting TNA. Furthermore, pepscan analysis suggested that the improvement in TNA was due to preservation of conformationally-sensitive, neutralizing epitopes because reactivity differences with the overlapping peptides did not adequately explain the dramatic improvement in TNA elicited by the abovementioned triple mutant. Due to the results presented in this dissertation, the RiVax triple mutant warrants further development as a ricin vaccine candidate.