Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA)

Abstract

Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design.

Keywords: Antibody; Epitope; Toxin; Vaccine.

Figure 1. Location of known B cell epitopes on RTA

Depiction of RTA highlighting the FDs and immunodominant regions described by O’Hara et al. [19]. Shown are the approximate locations of neutralizing (triangles) and non-neutralizing (circles) linear B cell epitopes.

Figure 2. RTA reactivity of mAbs

Reactivity of the neutralizing mAbs with RTA in different contexts was determined by ELISA; A) all mAbs and B) those that did not recognize ΔRTA. In panel A the mAbs are ordered from most potent mAb to least potent mAb from left to right of the graph based on data presented in Table 1. The cumulative OD₄₅₀ value (y-axis) was defined as the total reactivity of each form of RTA with (0.1 μg/well each in panel A and 1 μg/well each in panel B) with each mAb (0.5 μg/well; x-axis). RiVax is a rRTA with two point mutations; Y80A/V76M. rRTA* indicates that rRTA was coated on the plate at 1 μg/well. RiVax* indicates that RiVax was coated on the plate at 0.1 μg/well. Biotinylated ΔRTA was captured on the plate via an avidin bridge.

Figure 3. Western blot analysis of reactivity of select mAbs with ricin, RTA and RTB

The reactivity of MAbs (A) PH12, (B)TB12, (C)PA1 and (D) WECB2 with ricin holotoxin (H), RTA (A) and RTB (B) under non-denatured or denatured (boiled) and/or reduced conditions (β-mercaptoethanol;BME) was evaluated by Western Blot. All the mAbs recognized ricin holotoxin (filled arrowhead) and RTA but not RTB (open arrowhead) under all conditions tested.

Figure 4. Reactivity of mAbs with RTA point mutants

Reactivity of neutralizing mAbs with RTA point mutants was determined by ELISA. On the x-axis, the mAbs (0.5 μg/well) are ordered from most potent (left) to least potent (right). The cumulative OD₄₅₀ values (y-axis) are defined as the total reactivity of RTA, rRTA, or rRTA point mutants (1.0 μg/well) with each mAb (0.5 μg/well; x-axis). * indicates that RiVax coating was done at 0.1 μg/well.

Figure 5. Neutralizing B cell epitope clusters on RTA

PyMOL surface representation of ricin holotoxin depicting the four distinct clusters of neutralizing B cell epitopes on RTA that are described in this study. The clusters are color-coded consistent with Table 4. Ricin’s subunits, RTA and RTB, are colored grey and black, respectively. RTA’s active site is colored in magenta, and the cysteine residues that form the disulfide bond linking RTA to RTB are colored red.

Figure 6. Known and predicted B cell epitopes on RTA

PyMOL surface representation of ricin depicting known and predicted B cell epitopes on RTA. ElliPro and Discotope (available through the IEDB) were used to predict B cell epitopes on RTA. The colored shading corresponds to the following: dark orange, known epitopes but not predicted; marine blue, predicted and validated epitopes; light orange, predicted but not yet validated. Ricin’s subunits, RTA and RTB, are colored grey and black, respectively. RTA’s active site is colored in magenta, and the cysteine residues that form the disulfide bond linking RTA to RTB are colored red.