RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity.
Submitter: Shu-Ching Ou
Submission Date: Dec. 19, 2018, 10:31 a.m.
|Number of data points||84|
|Assays/Quantities/Protocols||Experimental Assay: Tm: CD ; Experimental Assay: Tm: TRP ; Experimental Assay: Tm: Light Scattering ; Experimental Assay: Tm1_apparent: DSC ; Experimental Assay: ΔH1_apparent: DSC ; Experimental Assay: Tm2_apparent: DSC ; Experimental Assay: ΔH2_apparent: DSC ; Derived Quantity: SD of Tm: CD ; Derived Quantity: SD of Tm: TRP ; Derived Quantity: SD of Tm: Light Scattering ; Derived Quantity: SD of Tm1_apparent: DSC ; Derived Quantity: SD of ΔH1_apparent: DSC ; Derived Quantity: SD of Tm2_apparent: DSC ; Derived Quantity: SD of ΔH2_apparent: DSC|
|Libraries||Variants for Ricin|