Unexpected synergistic HIV neutralization by a triple microbicide produced in rice endosperm.

The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency.

Downregulation of the CpSRP43 gene expression confers a truncated light-harvesting antenna (TLA) and enhances biomass and leaf-to-stem ratio in Nicotiana tabacum canopies.

MAIN CONCLUSION: Downregulation in the expression of the signal recognition particle 43 (SRP43) gene in tobacco conferred a truncated photosynthetic light-harvesting antenna (TLA property), and resulted in plants with a greater leaf-to-stem ratio, improved photosynthetic productivity and canopy biomass accumulation under high-density cultivation conditions. Evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a survival advantage for the organism in the wild, where sunlight is often the growth-limiting factor. In crop monocultures, however, this property is strongly counterproductive, when growth takes place under direct and excess sunlight. The large arrays of light-harvesting antennae in crop plants cause the surface of the canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis and forcing them to engage in wasteful dissipation of the excess energy. Evidence in this work showed that downregulation by RNA-interference approaches of the Nicotiana tabacum signal recognition particle 43 (SRP43), a nuclear gene encoding a chloroplast-localized component of the photosynthetic light-harvesting assembly pathway, caused a decrease in the light-harvesting antenna size of the photosystems, a corresponding increase in the photosynthetic productivity of chlorophyll in the leaves, and improved tobacco plant canopy biomass accumulation under high-density cultivation conditions. Importantly, the resulting TLA transgenic plants had a substantially greater leaf-to-stem biomass ratio, compared to those of the wild type, grown under identical agronomic conditions. The results are discussed in terms of the potential benefit that could accrue to agriculture upon application of the TLA-technology to crop plants, entailing higher density planting with plants having a greater biomass and leaf-to-stem ratio, translating into greater crop yields per plant with canopies in a novel agronomic configuration.

Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV.

Protein microbicides containing neutralizing antibodies and antiviral lectins may help to reduce the rate of infection with human immunodeficiency virus (HIV) if it is possible to manufacture the components in large quantities at a cost affordable in HIV-endemic regions such as sub-Saharan Africa. We expressed the antiviral lectin griffithsin (GRFT), which shows potent neutralizing activity against HIV, in the endosperm of transgenic rice plants (Oryza sativa), to determine whether rice can be used to produce inexpensive GRFT as a microbicide ingredient. The yield of (OS) GRFT in the best-performing plants was 223 µg/g dry seed weight. We also established a one-step purification protocol, achieving a recovery of 74% and a purity of 80%, which potentially could be developed into a larger-scale process to facilitate inexpensive downstream processing. (OS) GRFT bound to HIV glycans with similar efficiency to GRFT produced in Escherichia coli. Whole-cell assays using purified (OS) GRFT and infectivity assays using crude extracts of transgenic rice endosperm confirmed that both crude and pure (OS) GRFT showed potent activity against HIV and the crude extracts were not toxic towards human cell lines, suggesting they could be administered as a microbicide with only minimal processing. A freedom-to-operate analysis confirmed that GRFT produced in rice is suitable for commercial development, and an economic evaluation suggested that 1.8 kg/ha of pure GRFT could be produced from rice seeds. Our data therefore indicate that rice could be developed as an inexpensive production platform for GRFT as a microbicide component.

Rice endosperm produces an underglycosylated and potent form of the HIV-neutralizing monoclonal antibody 2G12.

Protein microbicides against HIV can help to prevent infection but they are required in large, repetitive doses. This makes current fermenter-based production systems prohibitively expensive. Plants are advantageous as production platforms because they offer a safe, economical and scalable alternative, and cereals such as rice are particularly attractive because they could allow pharmaceutical proteins to be produced economically and on a large scale in developing countries. Pharmaceutical proteins can also be stored as unprocessed seed, circumventing the need for a cold chain. Here, we report the development of transgenic rice plants expressing the HIV-neutralizing antibody 2G12 in the endosperm. Surprisingly for an antibody expressed in plants, the heavy chain was predominantly aglycosylated. Nevertheless, the heavy and light chains assembled into functional antibodies with more potent HIV-neutralizing activity than other plant-derived forms of 2G12 bearing typical high-mannose or plant complex-type glycans. Immunolocalization experiments showed that the assembled antibody accumulated predominantly in protein storage vacuoles but also induced the formation of novel, spherical storage compartments surrounded by ribosomes indicating that they originated from the endoplasmic reticulum. The comparison of wild-type and transgenic plants at the transcriptomic and proteomic levels indicated that endogenous genes related to starch biosynthesis were down-regulated in the endosperm of the transgenic plants, whereas genes encoding prolamin and glutaredoxin-C8 were up-regulated. Our data provide insight into factors that affect the functional efficacy of neutralizing antibodies in plants and the impact of recombinant proteins on endogenous gene expression.

Can the world afford to ignore biotechnology solutions that address food insecurity?

Genetically engineered (GE) crops can be used as part of a combined strategy to address food insecurity, which is defined as a lack of sustainable access to safe and nutritious food. In this article, we discuss the causes and consequences of food insecurity in the developing world, and the indirect economic impact on industrialized countries. We dissect the healthcare costs and lost productivity caused by food insecurity, and evaluate the relative merits of different intervention programs including supplementation, fortification and the deployment of GE crops with higher yields and enhanced nutritional properties. We provide clear evidence for the numerous potential benefits of GE crops, particularly for small-scale and subsistence farmers. GE crops with enhanced yields and nutritional properties constitute a vital component of any comprehensive strategy to tackle poverty, hunger and malnutrition in developing countries and thus reduce the global negative economic effects of food insecurity.

Low-dose interleukin 2 for the reduction of vascular inflammation in acute coronary syndromes (IVORY): protocol and study rationale for a randomised, double-blind, placebo-controlled, phase II clinical trial.

INTRODUCTION: Inflammation plays a critical role in the pathogenesis of atherosclerosis, the leading cause of ischaemic heart disease (IHD). Studies in preclinical models have demonstrated that an increase in regulatory T cells (Tregs), which have a potent immune modulatory action, led to a regression of atherosclerosis. The Low-dose InterLeukin 2 (IL-2) in patients with stable ischaemic heart disease and Acute Coronary Syndromes (LILACS) study, established the safety of low-dose IL-2 and its biological efficacy in IHD. The IVORY trial is designed to assess the effects of low-dose IL-2 on vascular inflammation in patients with acute coronary syndromes (ACS). METHODS AND ANALYSIS: In this study, we hypothesise that low-dose IL-2 will reduce vascular inflammation in patients presenting with ACS. This is a double-blind, randomised, placebo-controlled, phase II clinical trial. Patients will be recruited across two centres, a district general hospital and a tertiary cardiac centre in Cambridge, UK. Sixty patients with ACS (unstable angina, non-ST elevation myocardial infarction or ST elevation myocardial infarction) with high-sensitivity C reactive protein (hsCRP) levels >2 mg/L will be randomised to receive either 1.5×106 IU of low-dose IL-2 or placebo (1:1). Dosing will commence within 14 days of admission. Dosing will comprise of an induction and a maintenance phase. 2-Deoxy-2-[fluorine-18] fluoro-D-glucose (18F-FDG) positron emission tomography/CT (PET/CT) scans will be performed before and after dosing. The primary endpoint is the change in mean maximum target to background ratios (TBRmax) in the index vessel between baseline and follow-up scans. Changes in circulating T-cell subsets will be measured as secondary endpoints of the study. The safety and tolerability of extended dosing with low-dose IL-2 in patients with ACS will be evaluated throughout the study. ETHICS AND DISSEMINATION: The Health Research Authority and Health and Care Research Wales, UK (19/YH/0171), approved the study. Written informed consent is required to participate in the trial. The results will be reported through peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBER: NCT04241601.

Regulatory T-Cell Response to Low-Dose Interleukin-2 in Ischemic Heart Disease.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the artery wall. Regulatory T cells (Tregs) limit inflammation and promote tissue healing. Low doses of interleukin (IL)-2 have the potential to increase Tregs, but its use is contraindicated for patients with ischemic heart disease. METHODS: In this randomized, double-blind, placebo-controlled, dose-escalation trial, we tested low-dose subcutaneous aldesleukin (recombinant IL-2), given once daily for 5 consecutive days. In study part A, the primary end point was safety, and patients with stable ischemic heart disease were randomly assigned to receive placebo or to one of five dose groups (range, 0.3 to 3.0 × 106 IU daily). In study part B, patients with acute non-ST elevation myocardial infarction or unstable angina were randomly assigned to receive placebo or to one of two dose groups (1.5 and 2.5 × 106 IU daily). The coprimary end points were safety and the dose required to increase circulating Tregs by 75%. Single-cell RNA-sequencing of circulating immune cells was used to provide a mechanistic assessment of the effects of aldesleukin. RESULTS: Forty-four patients were randomly assigned to either study part A (n=26) or part B (n=18). In total, 3 patients withdrew before dosing, 27 received active treatment, and 14 received placebo. The majority of adverse events were mild. Two serious adverse events occurred, with one occurring after drug administration. In parts A and B, there was a dose-dependent increase in Tregs. In part B, the estimated dose to achieve a 75% increase in Tregs was 1.46 × 106 IU (95% confidence interval, 1.06 to 1.87). Single-cell RNA-sequencing demonstrated the engagement of distinct pathways and cell­cell interactions. CONCLUSIONS: In this phase 1b/2a study, low-dose IL-2 expanded Tregs without adverse events of major concern. Larger trials are needed to confirm the safety and to further evaluate the efficacy of low-dose IL-2 as an anti-inflammatory therapy for patients with ischemic heart disease. (Funded by the Medical Research Council, the British Heart Foundation, and others; ClinicalTrials.gov number, NCT03113773)

Investigating the Lowest Threshold of Vascular Benefits from LDL Cholesterol Lowering with a PCSK9 mAb Inhibitor (Alirocumab) in Patients with Stable Cardiovascular Disease (INTENSITY-HIGH): protocol and study rationale for a randomised, open label, parallel group, mechanistic study.

INTRODUCTION: Elevated low-density lipoprotein cholesterol (LDL-C) is a strong independent risk predictor of cardiovascular (CV) events, while interventions to reduce it remain the only evidence-based approach to reduce CV morbidity and mortality. Secondary prevention statin trials in combination with ezetimibe and/or proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors showed that there is no 'J shaped curve' in LDL-C levels with regard to CV outcomes. The lowest threshold beyond which reduction of LDL-C confers no further CV benefits has not been identified.The INTENSITY-HIGH study seeks to explore physiological mechanisms mediating CV benefits of LDL-C lowering by PCSK9 inhibition in patients with established cardiovascular disease (CVD). The study examines the changes in measures of endothelial function and vascular inflammation imaging following intervention with PCSK9 and against standard of care. METHODS AND ANALYSIS: This is a single-centre, randomised, open label, parallel group, mechanistic physiological study. It will include approximately 60 subjects with established CVD, with LDL-C of <4.1 mmol/L on high-intensity statins. All eligible participants will undergo 18-fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) scanning of the aorta and carotid arteries, as well as baseline endothelial function assessment. Subsequently, they will be randomised on a 1:1 basis to either alirocumab 150 mg or ezetimibe 10 mg/day. Repeat FDG-PET/CT scan and vascular assessments will be undertaken after 8 weeks of treatment. Any changes in these parameters will be correlated with changes in lipid levels and systemic inflammation biomarkers. ETHICS AND DISSEMINATION: The study received a favourable opinion from the Wales Research Ethics Committee 4, was registered on clinicaltrials.gov and conformed to International Conference for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Good Clinical Practice. The results of this study will be reported through peer-reviewed journals and conference presentations. TRIAL REGISTRATION NUMBER: NCT03355027.

Engineering metabolic pathways in plants by multigene transformation.

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.

The contribution of transgenic plants to better health through improved nutrition: opportunities and constraints.

Malnutrition is a prevalent and entrenched global socioeconomic challenge that reflects the combined impact of poverty, poor access to food, inefficient food distribution infrastructure, and an over-reliance on subsistence mono-agriculture. The dependence on staple cereals lacking many essential nutrients means that malnutrition is endemic in developing countries. Most individuals lack diverse diets and are therefore exposed to nutrient deficiencies. Plant biotechnology could play a major role in combating malnutrition through the engineering of nutritionally enhanced crops. In this article, we discuss different approaches that can enhance the nutritional content of staple crops by genetic engineering (GE) as well as the functionality and safety assessments required before nutritionally enhanced GE crops can be deployed in the field. We also consider major constraints that hinder the adoption of GE technology at different levels and suggest policies that could be adopted to accelerate the deployment of nutritionally enhanced GE crops within a multicomponent strategy to combat malnutrition.