A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

The impact of solvents on the toxicity of the banned parathion insecticide.

The aerial crop dusting and spraying of fields with the phosphorothioate insecticide parathion in the late 1900s, significantly improved crop yields but resulted in high levels of occupational toxicity in handlers and agricultural workers, as well as cases of intentional self-harm poisoning, culminating in its banning in many western countries by early 2000s. However because of the low solubility and volatility of parathion, most available products were formulated using organic solvents e.g. xylene, to increase the efficacy of the aerosols and dusts. In the present study, the toxicity of parathion was assessed when formulated in an aqueous solvents (ethanol/PBS (1:9)), and delivered to macaques as an aerosol. Doses of 780 µg/kg and 1.56 mg/kg were delivered one day apart, using a modified nebulizer calculated to result in lung deposition of ∼480 µg/kg with a similar or larger amount being swallowed; these doses being similar to the estimated lethal oral dose 286ug/kg - 1.43 mg/kg of formulated parathion in humans. Surprisingly, this dose (a combined amount of ∼14 mg) caused only low AChE inhibition and moderate BChE inhibition with no clinical symptoms, indicating that the use of organic solvents may have previously played a critical role in the severity of parathion toxicity following inhalation exposure. In addition, unlike constitutively toxic OPs, which are highly toxic when inhaled, these results are consistent with the idea that phosphorothioate insecticides appear to be more intoxicating following oral than inhalation exposure. However, this still remains uncertain because the presence of organic solvents in the ingested parathion studies was not always known.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Enhanced HIV SOSIP Envelope yields in plants through transient co-expression of peptidyl-prolyl isomerase B and calreticulin chaperones and ER targeting.

High yield production of recombinant HIV SOSIP envelope (Env) trimers has proven elusive as numerous disulfide bonds, proteolytic cleavage and extensive glycosylation pose high demands on the host cell machinery and stress imposed by accumulation of misfolded proteins may ultimately lead to cellular toxicity. The present study utilized the Nicotiana benthamiana/p19 (N.b./p19) transient plant system to assess co-expression of two ER master regulators and 5 chaperones, crucial in the folding process, to enhance yields of three Env SOSIPs, single chain BG505 SOSIP.664 gp140, CH505TF.6R.SOSIP.664.v4.1 and CH848-10.17-DT9. Phenotypic changes in leaves induced by SOSIP expression were employed to rapidly identify chaperone-assisted improvement in health and expression. Up to 15-fold increases were obtained by co-infiltration of peptidylprolvl isomerase (PPI) and calreticulin (CRT) which were further enhanced by addition of the ER-retrieval KDEL tags to the SOSIP genes; levels depending on individual SOSIP type, day of harvest and chaperone gene dosage. Results are consistent with reducing SOSIP misfolding and cellular stress due to increased exposure to the plant host cell's calnexin/calreticulin network and accelerating the rate-limiting cis-trans isomerization of Xaa-Pro peptide bonds respectively. Plant transient co-expression facilitates rapid identification of host cell factors and will be translatable to other complex glycoproteins and mammalian expression systems.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites.

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Introduction of the YTE mutation into the non-immunogenic HIV bnAb PGT121 induces anti-drug antibodies in macaques.

Recombinant antibodies play increasingly important roles as immunotherapeutic treatments for human cancers as well as inflammatory and infectious diseases and have revolutionized their management. In addition, their therapeutic potential may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains eg YTE (M252Y/S254T/T256E) and LS (M428L/N434S), as a consequence of increased half-lives and prolonged duration of protection. However, the functional properties of any biologic may be compromised by unanticipated immunogenicity in humans, rendering them ineffective. Several potent broadly neutralizing HIV monoclonal antibodies (bnAbs) have been identified that protect against SHIV challenge in macaque models and reduce HIV viremia in HIV-infected individuals. In the present study, the pharmacokinetics and immunogenicity of one or more 5mg/kg subcutaneous (SC) injections in naïve macaques of the HIV bnAb PGT121 and its PGT121-YTE mutant, both produced in plants, have been compared towards prolonging efficacy. Induction of anti-drug/anti-idiotypic antibodies (ADA, anti-id) has been monitored using both binding ELISAs and more functional inhibition of virus neutralization (ID50) assays. Timing of the anti-Id responses and their impact on pharmacokinetic profiles (clearance) and efficacy (protection) have also been assessed. The results indicate that ADA induction in naïve macaques may result both from injection of the previously non-immunogenic PGT121 into pre-primed animals and also by the introduction of the YTE mutation. Binding ADA antibody levels, induced in 7/10 macaques within two weeks of a first or second PGT121-YTE injection, were closely associated with both reduced pharmacokinetic profiles and loss of protection. However no correlation was observed with inhibitory ADA activity. These studies provide insights into both the structural features of bnAb and the immune status of the host which may contribute to the development of ADA in macaques and describe possible YTE-mediated changes in structure/orientation of HIV bnAbs that trigger such responses.