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.

Bilateral focused ultrasound medial thalamotomies for trigeminal neuropathic pain: a randomized controlled study.

OBJECTIVE: Medial thalamotomy has been shown to benefit patients with neuropathic pain, but widespread adoption of this procedure has been limited by reporting of clinical outcomes in studies without a control group. This study aimed to minimize confounders associated with medial thalamotomy for treating chronic pain by using modern MRI-guided stereotactic lesioning and a rigorous clinical design. METHODS: This prospective, double-blinded, randomized controlled trial in 10 patients with trigeminal neuropathic pain used sham procedures as controls. Participants underwent assessments by a pain psychologist and pain management clinician, including use of the following measures: the Numeric Pain Rating Scale (NPRS); patient-reported outcome measures; and patient's impression of improvement at baseline, 1 day, 1 week, 1 month, and 3 months postprocedure. Patients in the treated group underwent bilateral focused ultrasound (FUS) medial thalamotomy targeting the central lateral nucleus. Patients in the control group underwent sham procedures with energy output disabled. The primary efficacy outcome measure was between-group differences in pain intensity (using the NPRS) at baseline and at 3 months postprocedure. Adverse events were measured for safety and included MRI analysis. Exploratory measures of connectivity and metabolism were analyzed using diffusion tensor imaging, functional MRI, and PET, respectively. RESULTS: There were no serious complications from the FUS procedures. MRI confirmed bilateral medial thalamic ablations. There was no significant improvement in pain intensity from baseline to 3 months, either for patients undergoing FUS medial thalamotomy or for sham controls; and the between-group change in NPRS score as the primary efficacy outcome measure was not significantly different. Patient-reported outcome assessments demonstrated improvement (i.e., a decrease) only in pain interference with enjoyment of life at 3 months. There was a perception of benefit at 1 week, but only for patients treated with FUS and not for the sham cohort. Advanced neuroimaging showed that these medial thalamic lesions altered structural connectivity with the postcentral gyrus and demonstrated a trend toward hypometabolism in the insula and amygdala. CONCLUSIONS: This randomized controlled trial of bilateral FUS medial thalamotomy did not reduce the intensity of trigeminal neuropathic pain, although it should be noted that the ability to estimate the magnitude of treatment effects is limited by the small cohort.

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.

Protection of Newborn Macaques by Plant-Derived HIV Broadly Neutralizing Antibodies: a Model for Passive Immunotherapy during Breastfeeding.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIVSF162P3 was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 103 to 5 × 108 virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Aerosolized recombinant human butyrylcholinesterase delivered by a nebulizer provides long term protection against inhaled paraoxon in macaques.

The recent intentional use of nerve agents and pesticides in Europe and Afghanistan highlights the need for an effective countermeasure against organophosphates (OP) toxins. The most developed pretreatment candidate to date is plasma (native) human butyrylcholinesterase (HuBChE), which is limited in availability and because of its 1:1 stoichiometry with OPs, a large dose will present challenges when delivered parenterally both in terms of pharmacokinetics and manageability in the field. A tetrameric recombinant (r) form of human BChE produced in CHO-K1 cells with similar structure, in vivo stability and antidotal efficacy as the native form, has been developed to deliver rHuBChE as an aerosol (aer) to form a pulmonary bioshield capable of neutralizing inhaled OPs in situ and prevent AChE inhibition in the blood and in the brain; the latter associated with the symptoms of OP toxicity. Previous proof-of-concept macaque studies demonstrated that delivery of 9 mg/kg using a microsprayer inserted down the trachea, resulted in protection against an inhaled dose of 15ug/kg of aer-paraoxon (aer-Px) given 72 h later. In the present studies, pulmonary delivery of rHuBChE in macaques was achieved using Aerogen vibrating mesh nebulizers, similar to that used for human self-administration. The promising findings indicate that despite the poor lung deposition observed in macaques using nebulizers (13-20%), protective levels of RBC-AChE were still present in the blood even when exposure aer-Px (55 µg/kg) was delayed for five days. This long term retention of 5 mg/kg rHuBChE deposited in the lung bodes well for the use of an aer-rHuBChE pretreatment in humans where a user-friendly customized nebulizer with increased lung deposition up to 50% will provide even longer protection at a lower dose.

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.

Post-exposure treatment with the oxime RS194B rapidly reactivates and reverses advanced symptoms of lethal inhaled paraoxon in macaques.

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoxime e.g. pralidoxime, to reactivate the OP-inhibited AChE. However, commonly used oximes inefficiently cross the bloodbrain barrier and are rapidly cleared and their benefit is debated. Recent findings have demonstrated the ability of a novel zwitterionic, centrally acting, brain penetrating oxime (RS194B) to reverse severe symptoms and rapidly reactivate sarin-inhibited AChE in macaques, but it has not been tested following OP pesticide poisoning. In the present study, the symptoms following a lethal dose of inhaled paraoxon (100ug/kg), were shown to mimic those in insecticide poisoned individuals and were also rapidly reversed in macaques by post-exposure IM administration of 80mg/kg of RS194B. This occurred with a concomitant reactivation of AChE to 40-100% in<1hr and BChE (40% in 8h). These findings will be used to develop a macaque model with RS194B as a post-exposure treatment for insecticide poisoning and generate efficacy data for approval under the FDA Animal rule.

A highly stable minimally processed plant-derived recombinant acetylcholinesterase for nerve agent detection in adverse conditions.

Although recent innovations in transient plant systems have enabled gram quantities of proteins in 1-2 weeks, very few have been translated into applications due to technical challenges and high downstream processing costs. Here we report high-level production, using a Nicotiana benthamiana/p19 system, of an engineered recombinant human acetylcholinesterase (rAChE) that is highly stable in a minimally processed leaf extract. Lyophylized clarified extracts withstand prolonged storage at 70 °C and, upon reconstitution, can be used in several devices to detect organophosphate (OP) nerve agents and pesticides on surfaces ranging from 0 °C to 50 °C. The recent use of sarin in Syria highlights the urgent need for nerve agent detection and countermeasures necessary for preparedness and emergency responses. Bypassing cumbersome and expensive downstream processes has enabled us to fully exploit the speed, low cost and scalability of transient production systems resulting in the first successful implementation of plant-produced rAChE into a commercial biotechnology product.

Pharmacokinetics and immunogenicity of broadly neutralizing HIV monoclonal antibodies in macaques.

The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.

Rapid high-level production of functional HIV broadly neutralizing monoclonal antibodies in transient plant expression systems.

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production.

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.