Active immunity induced by passive IgG post-exposure protection against ricin.

Therapeutic antibodies can confer an instant protection against biothreat agents when administered. In this study, intact IgG and F(ab')2 from goat anti-ricin hyperimmune sera were compared for the protection against lethal ricin mediated intoxication. Similar ricin-binding affinities and neutralizing activities in vitro were observed between IgG and F(ab')2 when compared at the same molar concentration. In a murine ricin intoxication model, both IgG and F(ab')2 could rescue 100% of the mice by one dose (3 nmol) administration of antibodies 1 hour after 5 × LD50 ricin challenge. Nine days later, when the rescued mice received a second ricin challenge (5 × LD50), only the IgG-treated mice survived; the F(ab')2-treated mice did not. The experimental design excluded the possibility of residual goat IgG responsible for the protection against the second ricin challenge. Results confirmed that the active immunity against ricin in mice was induced quickly following the passive delivery of a single dose of goat IgG post-exposure. Furthermore, it was demonstrated that the induced active immunity against ricin in mice lasted at least 5 months. Therefore, passive IgG therapy not only provides immediate protection to the victim after ricin exposure, but also elicits an active immunity against ricin that subsequently results in long term protection.

Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 µ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D) of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

Role of the sodium hydrogen exchanger in maitotoxin-induced cell death in cultured rat cortical neurons.

Maitotoxin (MTX) is one of the most potent toxins known to date. It causes massive calcium (Ca(2+)) influx and necrotic cell death in various tissues. However, the exact mechanism(s) underlying its cellular toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca(2+) and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlue cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca(2+)](i) and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTX-induced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca(2+)](i), as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity.

A novel approach to development of monoclonal antibodies using native antigen for immunization and recombinant antigen for screening.

The production of monoclonal antibodies (MAb) specific to microbes is rapidly growing. Finding an appropriate antigen to screen hybridoma clones has become increasingly important. However, the conventional method, in which the purified antigen from the microbe is routinely used for screening, cannot avoid selection of false positive hybridoma clones, since even highly purified antigen is found to be contaminated with some other proteins from the microbe. In this study, MAbs against anthrax protective antigen (PA), the central component of the three-part toxin secreted by Bacillus anthracis were developed using a pair of the roughly purified native PA as an immunogen and the recombinant PA as a screening antigen without any possibility of false selection, since the recombinant PA was produced by a gene engineering approach and impossible to be contaminated with any other proteins from B. anthracis. In total, nine stable hybridoma clones secreting anti-PA MAbs were developed. All of them had the same type of heavy and light chains, IgG1/kappa. The binding profiles for these anti-PA MAbs were investigated by ELISA. This novel approach to the development of MAbs should be applicable to the production of MAbs to other microbes, especially to those from which antigens can hardly be purified to a high degree.

Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

Direct evidence of the existence of specific LPS binding sites on vascular endothelial cells.

OBJECTIVE: To investigate the binding characteristics of endothelial cell (EC) with LPS free from the participation of serum factors. METHODS: Laser confocal microscope was employed in the observation of the binding of EC with FITC-LPS. The KD and the binding sites of each EC were calculated by radioligand binding assay of receptors (RBA) using [(3)H]-LPS. RESULTS: The binding of EC with LPS was saturable, time and concentration dependent and it could be competed with overdosed LPS of the same type. The fluorescence mainly distributed in cytoplasm, especially near the nucleus, which could also be stained. CONCLUSIONS: There might be some specific LPS binding sites existing on ECs and LPS could function intracellularily.