Molecular responses of agroinfiltrated Nicotiana benthamiana leaves expressing suppressor of silencing P19 and influenza virus-like particles.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields.

Heterologous expression of influenza haemagglutinin leads to early and transient activation of the unfolded protein response in Nicotiana benthamiana.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress.

A genome-edited N. benthamiana line for industrial-scale production of recombinant glycoproteins with targeted N-glycosylation.

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker-free genome edited Nicotiana benthamiana N-glycosylation mutant (NbXF-KO) carrying inactivated β1,2-xylosyltransferase and α1,3-fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing.

Development and characterization of a plant-derived norovirus-like particle vaccine.

Background: Norovirus (NoV) is the most common cause of diarrheal episodes globally. Issues with in vitro cultivation systems, genetic variation, and animal models have hindered vaccine development. Plant-derived virus-like particles (VLPs) may address some of these concerns because they are highly immunogenic, can be administered by different routes, and can be rapidly produced to accommodate emerging viral strains.

An oligosaccharyltransferase from increases the N-glycan occupancy on plant-produced IgG1.

N-Glycosylation of immunoglobulin G1 (IgG1) at the heavy chain Fc domain (Asn297) plays an important role for antibody structure and effector functions. While numerous recombinant IgG1 antibodies have been successfully expressed in plants, they frequently display a considerable amount (up to 50%) of unglycosylated Fc domain. To overcome this limitation, we tested a single-subunit oligosaccharyltransferase from the protozoan (LdOST) for its ability to improve IgG1 Fc glycosylation.

Suitability of botanical extracts as components of complex mixtures used in herbal tea infusions-challenges and opportunities.

Herbal tea is a mainstay dosage form in practically all systems of traditional medicine and widely used in modern alternative and complementary medicine. Incorporating botanical extracts into herbal tea formulations is of vital interest to manufacturers as it allows for the use of herbal ingredients that would otherwise not be suitable for the dosage form, for instance, dosing requirements, solubility in water, sensory constraints Furthermore, reducing the amount of ingredients in a formula increases compliance with dosing recommendations and thus therapeutic benefit. However, formulating with botanical extracts comes with challenges, ranging from sourcing ingredients of appropriate quality, developing suitable methods for quality control with combinations of (herbal) ingredients, processing constraints such as hygroscopicity, solubility, dispersibility, homogeneity of distribution, and packaging machinability, all the way to stability required for hot-water infusion.

Durability and cross-reactivity of immune responses induced by a plant-based virus-like particle vaccine for COVID-19.

As the SARS-CoV-2 pandemic evolves, vaccine evaluation needs to include consideration of both durability and cross-reactivity. This report expands on previously reported results from a Phase 1 trial of an AS03-adjuvanted, plant-based coronavirus-like particle (CoVLP) displaying the spike (S) glycoprotein of the ancestral SARS-CoV-2 virus in healthy adults (NCT04450004). Humoral and cellular responses against the ancestral strain were evaluated 6 months post-second dose (D201) as secondary outcomes.

Safety and immunogenicity of an AS03-adjuvanted plant-based SARS-CoV-2 vaccine in Adults with and without Comorbidities.

The rapid spread of SARS-CoV-2 continues to impact humanity on a global scale with rising total morbidity and mortality. Despite the development of several effective vaccines, new products are needed to supply ongoing demand and to fight variants. We report herein a pre-specified interim analysis of the phase 2 portion of a Phase 2/3, randomized, placebo-controlled trial of a coronavirus virus-like particle (CoVLP) vaccine candidate, produced in plants that displays the SARS-CoV-2 spike glycoprotein, adjuvanted with AS03 (NCT04636697).

A Candidate Therapeutic Monoclonal Antibody Inhibits Both HRSV and HMPV Replication in Mice.

Human metapneumovirus (HMPV) and human respiratory virus (HRSV) are two leading causes of acute respiratory tract infection in young children. While there is no licensed drug against HMPV, the monoclonal antibody (mAb) Palivizumab is approved against HRSV for prophylaxis use only. Novel therapeutics against both viruses are therefore needed.

Regulation of Molecular Farming Products.

The regulation of molecular farming is a complex topic because plants and plant-based systems are relative newcomers among the many production platforms available for recombinant proteins. The regulations specific for different types of product (human/veterinary pharmaceuticals and medical devices, cosmetics, diagnostics, and research reagents) must therefore be overlaid with the regulations governing hitherto unfamiliar production platforms, and this must be achieved in different jurisdictions that handle genetically modified organisms (and genetically modified plants in particular) in very different ways. This chapter uses examples of different product types and production methods in three different jurisdictions (the USA, the EU, and Canada) to demonstrate some of the challenges facing the regulatory authorities.

Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine.

Background: Coronavirus-like particles (CoVLP) that are produced in plants and display the prefusion spike glycoprotein of the original strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are combined with an adjuvant (Adjuvant System 03 [AS03]) to form the candidate vaccine.

Methods: In this phase 3, multinational, randomized, placebo-controlled trial conducted at 85 centers, we assigned adults (≥18 years of age) in a 1:1 ratio to receive two intramuscular injections of the CoVLP+AS03 vaccine or placebo 21 days apart. The primary objective of the trial was to determine the efficacy of the CoVLP+AS03 vaccine in preventing symptomatic coronavirus disease 2019 (Covid-19) beginning at least 7 days after the second injection, with the analysis performed after the detection of at least 160 cases.

Elimination of receptor binding by influenza hemagglutinin improves vaccine-induced immunity.

The binding of influenza hemagglutinin (HA) to sialic acid (SA) receptors plays a well-defined role in shaping infection but the impact of such binding on vaccine responses has not yet been explored. We generated a virus-like particle (VLP) vaccine bearing the HA of H1N1 A/California/07/09 that is unable to bind to its α(2,6)-linked SA receptor (H1-VLP) and compared its immunogenicity and efficacy to a wild-type H1-VLP (H1-VLP) in mice. The H1-VLP elicited significantly stronger and more durable antibody responses (hemagglutination inhibition and microneutralization titers) and greater avidity maturation, likely attributable to improved germinal center formation.

Safety, immunogenicity, and protection provided by unadjuvanted and adjuvanted formulations of a recombinant plant-derived virus-like particle vaccine candidate for COVID-19 in nonhuman primates.

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018.

Phase 1 randomized trial of a plant-derived virus-like particle vaccine for COVID-19.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are being deployed, but the global need greatly exceeds the supply, and different formulations might be required for specific populations. Here we report Day 42 interim safety and immunogenicity data from an observer-blinded, dose escalation, randomized controlled study of a virus-like particle vaccine candidate produced in plants that displays the SARS-CoV-2 spike glycoprotein (CoVLP: NCT04450004 ). The co-primary outcomes were the short-term tolerability/safety and immunogenicity of CoVLP formulations assessed by neutralizing antibody (NAb) and cellular responses.

pH Gradient Mitigation in the Leaf Cell Secretory Pathway Attenuates the Defense Response of to Agroinfiltration.

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of infiltrated with the bacterial gene vector . An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector.

Production of Biopharmaceuticals in -Axillary Stem Growth as a Key Determinant of Total Protein Yield.

Data are scarce about the influence of basic cultural conditions on growth patterns and overall performance of plants used as heterologous production hosts for protein pharmaceuticals. Higher plants are complex organisms with young, mature, and senescing organs that show distinct metabolic backgrounds and differ in their ability to sustain foreign protein expression and accumulation. Here, we used the transient protein expression host as a model to map the accumulation profile of influenza virus hemagglutinin H1, a clinically promising vaccine antigen, at the whole plant scale.

Recombinant protein susceptibility to proteolysis in the plant cell secretory pathway is pH-dependent.

Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity-depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co-expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield.

Plant-produced biopharmaceuticals: A case of technical developments driving clinical deployment.

The ability to express heterologous proteins in plants has led to the concept of using plants as "bioreactors" or "biofactories" for the production of pharmaceutical proteins. Although initial studies were promising, the pathway to commercialization and deployment in a clinical setting has proven to be a somewhat rocky road. This Review examines the technical developments that have led to the current increase in interest in the use of plants for the production of pharmaceutical proteins, particularly in the context of clinical trials.

Cell wall biochemical alterations during Agrobacterium-mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco.

Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants.

Body Image and Anti-Fat Attitudes: An Experimental Study Using a Haptic Virtual Reality Environment to Replicate Human Touch.

It is well documented that anti-fat attitudes influence the interactions individuals have with overweight people. However, testing attitudes through self-report measures is challenging. In the present study, we explore the use of a haptic virtual reality environment to physically interact with overweight virtual human (VH).

Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues.