Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice

Abstract

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 two 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 (TNA) titers. Increased TNA values and seroconversion rates were evident at different antigen dosages and within seven days following the first booster. Quantitative stability/flexibility relationships (QSFR) analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98 to 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.

Influence of protein fold stability on immunogenicity and its implications for vaccine design

“…Immunization of mice with these derivatives revealed a 5–10-fold increased efficacy to induce toxin-neutralizing IgG antibodies [51]. …”

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

“…Toxin‐neutralizing antibodies do eventually appear, but they lag by several weeks and their appearance is not accompanied by a measurable increase in overall serum IgG . We propose that structure‐based computational modeling, similar to that employed here and in our previous studies, might be used to identify immunodominant epitopes on RTA that trigger the onset of high‐affinity, non‐neutralizing antibodies. Ablation of immunodominant non‐neutralizing epitopes through a process known as “resurfacing” would be expected to result in a dampening of non‐neutralizing antibody levels and a proportional increase in neutralizing titers following vaccination.…”

“…Hydrogen exchange studies were conducted with an inactivated mutant of RTA (V76M; Y80A), which is known as RiVax . The 2 point mutations do not alter the tertiary structure of RTA, as reported by Legler et al RiVax was expressed and purified as described in …”

Binding of the von Willebrand Factor A Domain of Capillary Morphogenesis Protein 2 to Anthrax Protective Antigen Vaccine Reduces Immunogenicity in Mice

“…For instance, efforts to improve the ricin vaccine (37,38), respiratory syncytial virus vaccine (24), or HIV-1 vaccine (39,40) have focused in part on improving the conformational stability to enhance the presentation of conformational epitopes (B-cell response) that have been shown to provide protective immunity. Immunogenicity in those cases is very likely tied to the flexibility of certain regions of the respective immunogenic proteins, with reduction in flexibility inducing stronger antibody binding (40).…”