Generation of Highly Efficient Equine-Derived Antibodies for Post-Exposure Treatment of Ricin Intoxications by Vaccination with Monomerized Ricin

Abstract

Ricin, a highly lethal toxin derived from the seeds of Ricinus communis (castor beans) is considered a potential biological threat agent due to its high availability, ease of production, and to the lack of any approved medical countermeasure against ricin exposures. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this work was to generate anti-ricin antitoxin that confers high level post-exposure protection against ricin challenge. Due to safety issues regarding the usage of ricin holotoxin as an antigen, we generated an inactivated toxin that would reduce health risks for both the immunizer and the immunized animal. To this end, a monomerized ricin antigen was constructed by reducing highly purified ricin to its monomeric constituents. Preliminary immunizing experiments in rabbits indicated that this monomerized antigen is as effective as the native toxin in terms of neutralizing antibody elicitation and protection of mice against lethal ricin challenges. Characterization of the monomerized antigen demonstrated that the irreversibly detached A and B subunits retain catalytic and lectin activity, respectively, implying that the monomerization process did not significantly affect their overall structure. Toxicity studies revealed that the monomerized ricin displayed a 250-fold decreased activity in a cell culture-based functionality test, while clinical signs were undetectable in mice injected with this antigen. Immunization of a horse with the monomerized toxin was highly effective in elicitation of high titers of neutralizing antibodies. Due to the increased potential of IgG-derived adverse events, anti-ricin F(ab’)2 antitoxin was produced. The F(ab’)2-based antitoxin conferred high protection to intranasally ricin-intoxicated mice; ~60% and ~34% survival, when administered 24 and 48 h post exposure to a lethal dose, respectively. In line with the enhanced protection, anti-inflammatory and anti-edematous effects were measured in the antitoxin treated mice, in comparison to mice that were intoxicated but not treated. Accordingly, this anti-ricin preparation is an excellent candidate for post exposure treatment of ricin intoxications.

Introduction to the Toxins Special Issue “Ricin Toxins”

“…Immunization of a horse with the monomerized toxin yielded high titers of neutralizing antibodies. Passive immunization of mice with equine derived F(ab') 2 based antitoxin conferred protection against a lethal intranasal ricin challenge [10].…”

“…The use of neutralizing antibodies is a promising post-exposure treatment against ricin intoxication. Falach et al [10] generated equine derived antibodies against ricin for post exposure treatment. They generated an inactivated toxin and constructed monomerized ricin antigen by irreversible reduction of the A and B subunits.…”

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

“…The toxin preparation stained using Coomassie blue and appeared in SDS‐ PAGE as two major bands of molecular masses of approximately 65 kDa (ricin toxin) and 120 kDa ( R. communis agglutinin). Purified ricin was prepared according to Falach et al…”

“…To select the most sensitive and specific peptides for ricin identification, tryptic digests of purified ricin (prepared according to Falach et al) were first analyzed by an Agilent 1200 Binary Pump system coupled to a QSTAR Elite Hybrid mass spectrometer (Applied Biosystems). This high resolution MS system enables the identification of ricin derived tryptic peptides, according to their accurate mass and sequence determination.…”

Post-Exposure Anti-Ricin Treatment Protects Swine against Lethal Systemic and Pulmonary Exposures

“…In the past decade, our group focused on pathogenesis and anti-ricin antibody-based protection studies in a small animal model, the mouse [8][9][10][11][12][13][14]. However, a large animal was required in order to establish a model that is more relevant to disease progression and post-exposure protection in humans.…”