Summary During the last decade, the spectre of an influenza pandemic of avian origin has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. The 2009 A/H1N1 (swine flu) outbreak has further emphasized the necessity to develop new solutions for pandemic influenza vaccines. Influenza virus‐like particles (VLPs)—non‐infectious particles resembling the influenza virus—represent a promising alternative to inactivated and split‐influenza virions as antigens, and they have shown uniqueness by inducing a potent immune response through both humoral and cellular components of the immune system. Our group has developed a plant‐based transient influenza VLP manufacturing platform capable of producing influenza VLPs with unprecedented speed. Influenza VLP expression and purification technologies were brought to large‐scale production of GMP‐grade material, and pre‐clinical studies have demonstrated that low doses of purified, plant‐produced influenza VLPs induce a strong and broad immune response in mice and ferrets. This review positions the recent developments towards the successful production of influenza VLPs in plants, including the production of VLPs from other human viruses and other forms of influenza antigens. The platform developed for large‐scale production of VLPs is also presented along with an assessment of the speed of the platform to produce the first experimental vaccine lots from the identification of a new influenza strain.

Summary A strain‐specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant‐based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high‐molecular‐weight structures. H5‐containing structures were purified and examination by transmission electron microscopy confirmed virus‐like particle (VLP) assembly. High‐performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP‐producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 µg of purified influenza H5‐VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 µg of H5‐VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

The recent swine H1N1 influenza outbreak demonstrated that egg-based vaccine manufacturing has an Achille's heel: its inability to provide a large number of doses quickly. Using a novel manufacturing platform based on transient expression of influenza surface glycoproteins in Nicotiana benthamiana, we have recently demonstrated that a candidate Virus-Like Particle (VLP) vaccine can be generated within 3 weeks of release of sequence information. Herein we report that alum-adjuvanted plant-made VLPs containing the hemagglutinin (HA) protein of H5N1 influenza (A/Indonesia/5/05) can induce cross-reactive antibodies in ferrets. Even low doses of this vaccine prevented pathology and reduced viral loads following heterotypic lethal challenge. We further report on safety and immunogenicity from a Phase I clinical study of the plant-made H5 VLP vaccine in healthy adults 18–60 years of age who received 2 doses 21 days apart of 5, 10 or 20 µg of alum-adjuvanted H5 VLP vaccine or placebo (alum). The vaccine was well tolerated at all doses. Adverse events (AE) were mild-to-moderate and self-limited. Pain at the injection site was the most frequent AE, reported in 70% of vaccinated subjects versus 50% of the placebo recipients. No allergic reactions were reported and the plant-made vaccine did not significantly increase the level of naturally occurring serum antibodies to plant-specific sugar moieties. The immunogenicity of the H5 VLP vaccine was evaluated by Hemagglutination-Inhibition (HI), Single Radial Hemolysis (SRH) and MicroNeutralisation (MN). Results from these three assays were highly correlated and showed similar trends across doses. There was a clear dose-response in all measures of immunogenicity and almost 96% of those in the higher dose groups (2×10 or 20 µg) mounted detectable MN responses. Evidence of striking cross-protection in ferrets combined with a good safety profile and promising immunogenicity in humans suggest that plant-based VLP vaccines should be further evaluated for use in pre-pandemic or pandemic situations.Trial RegistrationClinicalTrials.gov NCT00984945

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SummaryPlant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N -glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human β 1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N -glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.

Understanding the mechanisms controlling crop effects on soil aggregation is necessary to develop sustainable soil management practices. Changes in soil aggregation, carbohydrates, and fungal vs. bacterial biomass were monitored following a shift from fallow (bare soil) to various cropping systems. Faba bean (Vicia faba L.) and wheat (Triticum aestivum L.) were used as annual cropping systems, while alfalfa (Medicago sativa L.), timothy (Phleum pratense L.), bromegrass (Bromus inermis L.), and reed canarygrass (Phalaris arundinacea L.) were used as perennial cropping systems. Crops were established in spring 1989, on a silty clay loam (fine, mixed, frigid Typic Dystro‐chrept) and a clay loam (fine‐loamy, mixed, frigid Typic Humaquept) near Québec City (Canada). After three growing seasons, the mean‐weight diameter of water‐stable aggregates (MWD) was higher under reed canarygrass (2.26 mm in the clay loam and 2.45 mm in the silty clay loam) and timothy (2.13 and 2.26 mm) than under faba bean (1.92 and 1.89 mm) or wheat (2.06 and 1.57 mm). Intermediate values were found under alfalfa and bromegrass. Changes in MWD were not correlated with microbial biomass C. Acid‐hydrolyzable carbohydrates correlated with MWD in the silty clay loam (r2 = 0.42**) but the relationship decreased with higher carbohydrate levels in the clay loam (r2 = 0.05). Close correlations were found between MWD and both fungal glucosamine (r2 = 0.68***, soils combined) and bacterial muramic acid (r2 = 0.48***). Changes in MWD were mostly reflected in aggregates >2 mm, and the close relationship with fungal glucosamine suggests that fungi played a dominant role in soil macroaggregation. Measurement of muramic acid and glucosamine is proposed as a potential approach to compare bacterial vs. fungal contributions to soil aggregation.

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SummaryProteolytic degradation represents a significant barrier to the efficient production of several recombinant proteins in plants, both in vivo during their expression and in vitro during their recovery from source tissues. Here, we describe a strategy to protect recombinant proteins during the recovery process, based on the coexpression of a heterologous proteinase inhibitor acting as a 'mouse trap' against the host proteases during extraction. After