RESUMEN
Ricin, a plant-derived toxin originating from the seeds of Ricinus communis (castor bean plant), is one of the most lethal toxins known. To date, there is no approved post-exposure therapy for ricin exposures. This work demonstrates for the first time the therapeutic efficacy of equine-derived anti-ricin F(ab')2 antibodies against lethal pulmonary and systemic ricin exposures in swine. While administration of the antitoxin at 18 h post-exposure protected more than 80% of both intratracheally and intramuscularly ricin-intoxicated swine, treatment at 24 h post-exposure protected 58% of the intramuscular-exposed swine, as opposed to 26% of the intratracheally exposed animals. Quantitation of the anti-ricin neutralizing units in the anti-toxin preparations confirmed that the disparate protection conferred to swine subjected to the two routes of exposure stems from variance between the two models. Furthermore, dose response experiments showed that approximately 3 times lesser amounts of antibody are needed for high-level protection of the intramuscularly compared to the intratracheally intoxicated swine. This study, which demonstrates the high-level post-exposure efficacy of anti-ricin antitoxin at clinically relevant time-points in a large animal model, can serve as the basis for the formulation of post-exposure countermeasures against ricin poisoning in humans.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antitoxinas/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Ricina/antagonistas & inhibidores , Administración por Inhalación , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Caballos , Inyecciones Intramusculares , Ratones , Ricina/administración & dosificación , Ricina/inmunología , Ricina/envenenamiento , Sus scrofa , Factores de TiempoRESUMEN
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.
Asunto(s)
Antígenos/toxicidad , Antitoxinas/uso terapéutico , Ricina/toxicidad , Animales , Anticuerpos Neutralizantes/inmunología , Antígenos/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Femenino , Caballos , Ratones , Conejos , Ricina/inmunología , VacunaciónRESUMEN
UNLABELLED: Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus ostreatus vp1 expression occurred in Mn(2+)-sufficient medium. This seems to be a biological contradiction. The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn(2+)-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates, Mn(2+), Orange II (OII), and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-mediated oxidation conditions, VP1 was able to cleave the azo bond only in asymmetric mode, while under the optimum conditions for direct oxidation (absence of Mn(2+) at pH 3) both symmetric and asymmetric cleavages occurred. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture. IMPORTANCE: VP1 is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays a fundamental role in biodegradation. This enzyme exhibits a versatile nature, as it can oxidize different substrates under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites that are responsible for direct oxidation of various aromatic compounds, including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1, which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but they perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for the potential utilization of P. ostreatus and other white rot fungi.