Feral Hogs Management at Merritt Island National Wildlife Refuge: Analysis of Current Management Program

This ST1 Technical Memorandum (TM) summarizes a two-month project on feral hog management in Merritt Island National Wildlife Refuge (MINWR). For this project, feral hogs were marked and recaptured, with the help of local trappers, to estimate population size and habitat preferences. Habitat covers included vegetation cover and Light Detection and Ranging (LIDAR) data for MINWR. In addition, an analysis was done of hunting records compiled by the Refuge and hog-car accidents compiled by KSC Security

Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication

<i>In vivo</i> LeTx neutralization.

<p>Rats (n≥6 for each group) were i.m. administered with the indicated doses of (A) cAb 29 or (B) mAb 29, followed by i.v. challenge with LeTx (20 µg PA and 10 µg LF), 17 hours later. Animal survival was monitored for the next 24 hours.</p

<i>In vitro</i> LeTx neutralization.

<p>Toxin complex (5 µg/ml PA and 2 µg/ml LF) was pre-incubated for one hour with increasing concentrations of cAb 29 (triangles) or mAb 29 (squares), and added to J774A.1 mouse macrophage cells for 5 hours. Cell survival was then determined by MTT, and was plotted as percent of untreated control cells. Points are mean±STD of triplicate determinants.</p

Binding kinetics of the PA-specific antibodies.

a<p>Binding kinetics values derived from global curve-fitting analysis using divalent analysis model.</p>b<p>Binding kinetics values derived from global curve-fitting analysis using 1∶1 Langmuir binding model.</p

Kinetic analysis of mAb 29 and cAb 29 binding to PA.

<p>SPR sensograms obtained during injection of (A) 100–2000 nM of PA on anti-human captured cAb 29 (∼300 RU), refers to the direct format; or (B) 5–100 nM of cAb 29 on immobilized PA (∼900 RU), refers to the indirect format. Similar sensograms were obtained for the mAb 29 (results not shown).</p

Kinetics of anti-PA and PA-neutralizing antibody development.

<p>Average titer values of total anti-PA ELISA antibodies (filled circles) of eight immunized mice and LeTx neutralizing antibody titers (empty circles) of mice 3, 4, 5 and 7, throughout the immunization process. Arrows indicate PA-immunization time points; Data points are mean±STD.</p

Protection of Guinea pigs from anthrax infection by antibody administration.

a<p>Animals were challenged with s.c administration of 40LD₅₀ of <i>B. anthracis</i> Vollum spores.</p>b<p>Animals that survived the 1^st challenge were re-infected with 40LD₅₀ of <i>B. anthracis</i> Vollum spores.</p>C<p>One animal was omitted from re-challenged, due to bites injury.</p