Medicago (Canada)

<title>Abstract</title> In this paper, we present simulation studies of scintillation effects and tracking of global navigation satellite system (GNSS)-like signals propagating from a low Earth orbit (LEO) satellite through ionospheric plasma irregularities to a receiver on the Earth’s surface. Realistic strong scintillation effects are simulated by propagating signals through a phase screen based on real GNSS scintillation signals. Configurations with different phase screen altitudes, LEO satellite orbits, and scintillation severity levels are simulated. We first statistically analyze the number of fades, fading depth and duration, and phase rate disturbance in the signal. A software-defined radio receiver is then applied to process the simulated signals. The impacts of such signals on tracking performance, e.g., <italic>C</italic>/<italic>N</italic>₀, cycle slip, and loss of lock, are analyzed and compared with that of the same signal traveling through the same irregularity, but from a medium Earth orbit. The results provide a quantitative assessment of the effect of ionospheric plasma irregularities on LEO satellite signal transmissions and receiver processing.

The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the Spike (S) protein of Severe Acute Respiratory Syndrome Coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana. Relying on bacterial vector Agrobacterium tumefaciens, this process is favored by the co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We previously characterized effects of influenza virus hemagglutinin forming VLPs through similar processes. However, leaf samples were only collected after six days of expression and it remains unknown whether influenza VLPs and CoVLPs induce similar responses. Here, time course sampling was used to profile responses of N. benthamiana leaf cells expressing P19 only, or P19 along with the S protein. The latter triggered early, but transient activation of the unfolded protein response and waves of transcription factor genes involved in immunity. Accordingly, defense genes were induced with different expression kinetics, including those promoting lignification, terpene biosynthesis, and oxidative stress. Crosstalk between stress hormone pathways also occurred, notably leading to the repression of jasmonic acid biosynthesis genes after agroinfiltration, and dampening of salicylic acid-inducible responses upon S protein accumulation. Overall, influenza VLP- and CoVLP-induced responses broadly overlapped, suggesting nanoparticle production to have the most effects on plant immunity, regardless of the virus surface proteins expressed. Taking advantage of RNAseq inferences, we finally show the co-expression of Kunitz trypsin inhibitors to reduce CoVLP-induced defense and leaf symptoms, with no adverse effect on plant productivity.

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. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Cell-mediated immunity is an important component of immediate and long-term anti-viral protection. Dendritic cells (DCs) are essential for the induction of cell-mediated immunity by instructing the activation and differentiation of antigen-specific T cell responses. Activated DCs that express co-stimulatory molecules and pro-inflammatory cytokines are necessary to promote the development of type 1 immune responses required for viral control. Here we report that plant-derived virus-like particles (VLPs) bearing influenza hemagglutinins (HA) directly stimulate mouse and human DCs. DCs exposed to H1- and, to a lesser extent, H5-VLPs in vitro rapidly express co-stimulatory molecules and produce pro-inflammatory cytokines including IL-12, IL-6 and TNFα. Furthermore, these VLPs support the activation and differentiation of antigen-specific T cell responses. Mechanistically, H1-VLPs stimulate the activation of kinases typically activated downstream of pattern recognition receptors including AKT, p38, and p42/44 ERK. In vivo, immunization with plant-derived VLPs induce the accumulation of both cDC1s and cDC2 in the draining lymph node and a corresponding increase in T and B cells. VLPs devoid of HA protein activate DCs, suggesting they are intrinsically immunostimulatory. Together, the results demonstrate that these candidate plant-derived VLP vaccines have an inherent and direct stimulatory effect on DCs and can enhance the ability of DCs to promote Type 1 immune responses.

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A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.

In 1986, it was shown that tobacco plants and sunflower calluses could express recombinant human growth hormone as a fusion protein [1]. Since then, a diverse range of plant systems has been used for the production of pharmaceuticals [2, 3]. We have developed a production system based on the leaves of alfalfa (Medicago sativa L.), a choice made originally because of the plant’s many favorable agronomic characteristics. Alfalfa is a perennial plant, so vegetative growth can be maintained for many years. For molecular farming, this characteristic, combined with the ease of clonal propagation through stem cutting, makes alfalfa a robust bioreactor with regard to batch-to-batch reproducibility. Among perennial plants, legume forage crops such as alfalfa have the advantage of fixing atmospheric nitrogen, thus reducing the need for fertilizers. Moreover, as a feed fodder crop, alfalfa has benefited from important research aiming to increase leaf protein content, so that today’s varieties produce as much as 30 mg total protein per gram fresh weight. In addition to these appealing agronomic characteristics, biotechnological research has revealed additional benefits for the production of pharmaceuticals in alfalfa. Expression cassettes have been optimized for protein expression in alfalfa leaves. Methods for transient protein expression have been developed so that it is now possible to use agroinfiltration or the transformation of protoplasts for earlystage demonstration and validation steps. In addition, glycosylation studies have shown that alfalfa is capable of producing recombinant glycoproteins with homogenous (uniform) glycosylation patterns. This chapter provides an overview of the tools that have been developed and optimized specifically for the production of pharmaceuticals in alfalfa, with the emphasis on recent technological breakthroughs. The ability of alfalfa leaves to produce complex recombinant proteins of pharmaceutical interest is discussed and illustrated with recent data obtained in our laboratories. Data are presented concerning the production and characterization of alfalfa-derived C5-1, a diagnostic anti-human 1