The heme binding protein ChuX is a regulator of heme degradation by the ChuS protein in Escherichia coli O157:H7.

Escherichia coli O157:H7 possesses an 8-gene cluster (chu genes) that contains genes involved in heme transport and processing from the human host. Among the chu genes, four encode cytoplasmic proteins (ChuS, ChuX, ChuY and ChuW). ChuX was previously shown to be a heme binding protein and to assist ChuW in heme degradation under anaerobic conditions. The purpose of this work was to investigate if ChuX works in concert with ChuS, which is a protein able to degrade heme by a non-canonical mechanism and release the iron from the porphyrin under aerobic conditions using hydrogen peroxide as the oxidant. We showed that when the heme-bound ChuX and apo-ChuS protein are mixed, heme is efficiently transferred from ChuX to ChuS. Heme-bound ChuX displayed a peroxidase activity with ABTS and H2O2 but not heme-bound ChuS, which is an efficient test to determine the protein to which heme is bound in the ChuS-ChuX complex. We found that ChuX protects heme from chemical oxidation and that it has no heme degradation activity by itself. Unexpectedly, we found that ChuX inhibits heme degradation by ChuS and stops the reaction at an early intermediate. We determined using surface plasmon resonance that ChuX interacts with ChuS and that it forms a relatively stable complex. These results indicate that ChuX in addition to its heme transfer activity is a regulator of ChuS activity, a function that was not described before for any of the heme carrier protein that delivers heme to heme degradation enzymes.

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. METHODS: NoV VLPs (NoVLP) composed of the surface viral protein (VP) 1 of the GI and GII genogroups were produced in Nicotiana benthamiana using an Agrobacterium tumefaciens-based recombinant transient expression system. Leaves from infiltrated plants were harvested and NoVLPs were extracted and purified. The safety and immunogenicity of the GII.4 NoVLP, the genotype currently causing most human disease, were subsequently examined in rabbits and mice. RESULTS: Fifteen GI and GII NoVLPs were successfully expressed in N. benthamiana and were structurally similar to NoV virions, as determined by cryogenic transmission electron microscopy. The NoVLP was well-tolerated, with no local or systemic signs of toxicity in rabbits. Three intramuscular doses of the GII.4 NoVLP adjuvanted with aluminum hydroxide induced robust IgG titers, IgG-secreting cells, histo-blood group antigen blocking titers, and IFNγ-secreting T cells in mice. In addition to circulating antibodies, oral administration of the NoVLP in mice induced significant IgA levels in feces, indicative of a mucosal response. CONCLUSIONS: The plant-made NoVLP vaccine was safe and immunogenic in mice and rabbits. Multi-modal vaccination, combining oral and intramuscular administration could be considered for future clinical development to maximize systemic and mucosal immune responses.

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. RESULTS: A total of 24,141 volunteers participated in the trial; the median age of the participants was 29 years. Covid-19 was confirmed by polymerase-chain-reaction assay in 165 participants in the intention-to-treat population; all viral samples that could be sequenced contained variants of the original strain. Vaccine efficacy was 69.5% (95% confidence interval [CI], 56.7 to 78.8) against any symptomatic Covid-19 caused by five variants that were identified by sequencing. In a post hoc analysis, vaccine efficacy was 78.8% (95% CI, 55.8 to 90.8) against moderate-to-severe disease and 74.0% (95% CI, 62.1 to 82.5) among the participants who were seronegative at baseline. No severe cases of Covid-19 occurred in the vaccine group, in which the median viral load for breakthrough cases was lower than that in the placebo group by a factor of more than 100. Solicited adverse events were mostly mild or moderate and transient and were more frequent in the vaccine group than in the placebo group; local adverse events occurred in 92.3% and 45.5% of participants, respectively, and systemic adverse events in 87.3% and 65.0%. The incidence of unsolicited adverse events was similar in the two groups up to 21 days after each dose (22.7% and 20.4%) and from day 43 through day 201 (4.2% and 4.0%). CONCLUSIONS: The CoVLP+AS03 vaccine was effective in preventing Covid-19 caused by a spectrum of variants, with efficacy ranging from 69.5% against symptomatic infection to 78.8% against moderate-to-severe disease. (Funded by Medicago; ClinicalTrials.gov number, NCT04636697.).

Safety and immunogenicity of a plant-derived rotavirus-like particle vaccine in adults, toddlers and infants.

BACKGROUND: This study is the first clinical trial for a parenteral non-replicating rotavirus vaccine developed using virus-like particle (VLP) technology. METHODS: This open-labeled, randomized, placebo-controlled trial was conducted in two parts: Part A (a first-in-human study in Australian adults) and Part B (ascending dose and descending age in South African adults, toddlers and infants). In Part A, two cohorts of 10 adults were assigned to receive a single intramuscular injection of 1 of 2 escalating dose levels of the rotavirus VLP (Ro-VLP) vaccine (7 µg or 21 µg) or placebo. In Part B, one cohort of 10 adults was assigned to receive a single injection of the Ro-VLP vaccine (21 µg) or placebo, two cohorts of 10 toddlers were assigned to receive 2 injections of 1 of 2 escalating dose levels of the Ro-VLP vaccine (7 µg or 21 µg) or placebo 28 days apart, and three cohorts of 20 infants were assigned to receive 3 injections of 1 of 3 escalating dose levels of the Ro-VLP vaccine (2.5 µg, 7 µg or 21 µg) or placebo or 2 doses of oral Rotarix 28 days apart. Safety, reactogenicity and immunogenicity were assessed. RESULTS: There were no safety or tolerability concerns after administration of the Ro-VLP vaccine. The Ro-VLP vaccine induced an anti-G1P[8] IgG response in infants 4 weeks after the second and third doses. Neutralizing antibody responses against homologous G1P[8] rotavirus were higher in all Ro-VLP infant groups than in the placebo group 4 weeks after the third dose. No heterotypic immunity was elicited by the Ro-VLP vaccine. CONCLUSIONS: The Ro-VLP vaccine was well tolerated and induced a homotypic immune response in infants, suggesting that this technology platform is a favorable approach for a parenteral non-replicating rotavirus vaccine. CLINICAL TRIAL REGISTRATION: NCT03507738.

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

BACKGROUND: Virus-like particles (VLPs) are unable to replicate in the recipient but stimulate the immune system through recognition of repetitive subunits. Parenterally delivered rotavirus-VLP (Ro-VLP) vaccine could have the potential to overcome the weaknesses of licensed oral live-attenuated rotavirus vaccines, namely, low efficacy in low-income and high mortality settings and a potential risk of intussusception. METHODS: A monovalent Ro-VLP composed of viral protein (VP) 7, VP6 and VP2 of G1 genotype specificity was produced in Nicotiana benthamiana using Agrobacterium tumefaciens infiltration-based transient recombinant expression system. Plants expressing recombinant G1 Ro-VLP were harvested, then the resultant biomass was processed through a series of clarification and purification steps including standard extraction, filtration, ultrafiltration and chromatography. The purified G1 Ro-VLP was subsequently examined for its immunogenicity and toxicological profile using animal models. RESULTS: G1 Ro-VLP had a purity of ≥90% and was structurally similar to triple-layered rotavirus particles as determined by cryogenic transmission electron microscopy. Two doses of aluminum hydroxide-adjuvanted G1 Ro-VLP (1 µg, 5 µg or 30 µg), administered intramuscularly, elicited a robust homotypic neutralizing antibody response in rats. Also, rabbits administered G1 Ro-VLP (10 µg or 30 µg) four times intramuscularly with aluminum hydroxide adjuvant did not show any significant toxicity. CONCLUSIONS: Plant-derived Ro-VLP composed of VP7, VP6 and VP2 structural proteins would be a plausible alternative to live-attenuated oral rotavirus vaccines currently distributed worldwide.

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. Secondary outcomes in this ongoing study include safety and immunogenicity assessments up to 12 months after vaccination. Adults (18-55 years, n = 180) were randomized at two sites in Quebec, Canada, to receive two intramuscular doses of CoVLP (3.75 µg, 7.5 µg, and 15 µg) 21 d apart, alone or adjuvanted with AS03 or CpG1018. All formulations were well tolerated, and adverse events after vaccination were generally mild to moderate, transient and highest in the adjuvanted groups. There was no CoVLP dose effect on serum NAbs, but titers increased significantly with both adjuvants. After the second dose, NAbs in the CoVLP + AS03 groups were more than tenfold higher than titers in Coronavirus 2019 convalescent sera. Both spike protein-specific interferon-γ and interleukin-4 cellular responses were also induced. This pre-specified interim analysis supports further evaluation of the CoVLP vaccine candidate.

Effects of Hexane on Protein Profile, Solubility and Foaming Properties of Defatted Proteins Extracted from Tenebrio molitor Larvae.

Inclusion of edible insects in human diets is increasingly promoted as a sustainable source of proteins with high nutritional value. While consumer acceptability remains the main challenge to their integration into Western food culture, the use of edible insects as meal and protein concentrate could decrease neophobia. The defatting of edible insects, mostly done with hexane, is the first step in producing protein ingredients. However, its impact on protein profiles and techno-functionality is still unclear. Consequently, this study compares the protein profiles of hexane-defatted and non-hexane-defatted yellow mealworm (Tenebrio molitor) meals and protein extracts, and evaluates the impact of hexane on protein solubility and foaming properties. Results showed that profiles for major proteins were similar between hexane-defatted and non-defatted samples, however some specific content differences (e.g., hexamerin 2) were observed and characterized using proteomic tools. Protein solubility was markedly lower for T. molitor meals compared to protein extracts. A large increase in the foaming capacity was observed for defatted fractions, whereas foam stability decreased similarly in all fractions. Consequently, although the hexane-defatting step was largely studied to produce edible insect protein ingredients, it is necessary to precisely understand its impact on their techno-functional properties for the development of food formulations.

Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism.

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.

Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations.

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen-oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a "sliding" conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

Resonance Raman studies on the flavohemoglobin of the protist Giardia intestinalis: evidence of a type I/II-peroxidase-like heme environment and roles of the active site distal residues.

Flavohemoglobins are microbial enzymes that counter nitrosative stress, but the details of their underlying enzymatic activities and structure-function relationships are not completely understood. These enzymes have been identified in Gram-negative bacteria, certain fungi, and the parasitic protist Giardia intestinalis (gFlHb) which, despite lacking the ability to make heme, encodes several hemeproteins. To gain knowledge about the biophysical properties of the active site of gFlHb, we used resonance Raman spectroscopy to probe the wild-type protein and variants at globin domain positions E11, E7, and B10 on the distal, ligand-binding side of the heme. The heme of gFlHb has a peroxidase-like environment resembling that of the well-characterized E. coli flavohemoglobin HMP. We provide evidence that gFlHb has two Fe-His stretching modes, a feature that also occurs in type I/II-peroxidases in which a proximal histidine with strong imidazolate character and a nearby carboxylic acid residue can exist as a tautomeric pair depending on the position of a shared proton. Characterization of the distal variants Tyr30Phe, Gln54Leu, and Leu59Ala shows that TyrB10 and GlnE7 but not LeuE11 are implicated in stabilisation of bound exogenous ligands such as CO and O2. Our work revealed that several biophysical properties of the heme active site of gFlHb are highly conserved compared to HMP and suggest that they are conserved across the flavohemoglobin family.

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. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.

Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases.

The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to the buildup of an intermediate species with a Soret band at ∼435 nm (I435). The nature of this intermediate is, however, debated. On the one hand, I435 has been presented as a compound II species that can be photoactivated to compound I. A competing alternative involves the assignment of I435 to a ferric-nitrosyl species. Similar to cytochromes P450, the buildup of I435 occurs in nitric oxide synthases (NOSs) upon their reaction with excess PN. Interestingly, the NOS isoforms vary in their capacity to detoxify/activate PN, although they all show the buildup of I435. To better understand PN activation/detoxification by heme proteins, a definitive assignment of I435 is needed. Here we used a combination of fine kinetic analysis under specific conditions (pH, PN concentrations, and PN/NOSs ratios) to probe the formation of I435. These studies revealed that I435 is not formed upon homolytic cleavage of the O-O bond of PN, but instead arises from side reactions associated with excess PN. Characterization of I435 by resonance Raman spectroscopy allowed its identification as a ferric iron-nitrosyl complex. Our study indicates that the model used so far to depict PN interactions with hemo-thiolate proteins, i.e., leading to the formation and accumulation of compound II, needs to be reconsidered.

Oxygen activation in NO synthases: evidence for a direct role of the substrate.

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho-physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast-kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise Fe(II)-O2 reaction intermediates in the presence of L-arginine (Arg), alkyl- and aryl-guanidines. The presence of new reaction intermediates, such as ferric haem-peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H-bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (N(ω)-hydroxy-L-arginine oxidation).

An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III).

As part of the machinery to acquire, internalize and utilize heme as a source of iron from the host, some bacteria possess a canonical heme oxygenase, where heme plays the dual role of substrate and cofactor, the later catalyzing the cleavage of the heme moiety using O2 and electrons, and resulting in biliverdin, carbon monoxide and ferrous non-heme iron. We have previously reported that the Escherichia coli O157:H7 ChuS protein, which is not homologous to heme oxygenases, can bind and degrade heme in a reaction that releases carbon monoxide. Here, we have pursued a detailed characterization of such heme degradation reaction using stopped-flow UV-visible absorption spectrometry, the characterization of the intermediate species formed in such reaction by EPR spectroscopy and the identification of reaction products by NMR spectroscopy and Mass spectrometry. We show that hydrogen peroxide (in molar equivalent) is the key player in the degradation reaction, at variance to canonical heme oxygenases. While the initial intermediates of the reaction of ChuS with hydrogen peroxide (a ferrous keto π neutral radical and ferric verdoheme, both identified by EPR spectroscopy) are in common with heme oxygenases, a further and unprecedented reaction step, involving the cleavage of the porphyrin ring at adjacent meso-carbons, results in the release of hematinic acid (a monopyrrole moiety identified by NMR spectroscopy), a tripyrrole product (identified by Mass spectrometry) and non-heme iron in the ferric oxidation state (identified by EPR spectroscopy). Overall, the unprecedented reaction of E. coli O157:H7 ChuS provides evidence for a novel heme degradation activity in a Gram-negative bacterium.

Flavoenzyme CrmK-mediated substrate recycling in caerulomycin biosynthesis.

Substrate salvage or recycling is common and important for primary metabolism in cells but is rare in secondary metabolism. Herein we report flavoenzyme CrmK-mediated shunt product recycling in the biosynthesis of caerulomycin A (CRM A 1), a 2,2'-bipyridine-containing natural product that is under development as a potent novel immunosuppressive agent. We demonstrated that the alcohol oxidase CrmK, belonging to the family of bicovalent FAD-binding flavoproteins, catalyzed the conversion of an alcohol into a carboxylate via an aldehyde. The CrmK-mediated reactions were not en route to 1 biosynthesis but played an unexpectedly important role by recycling shunt products back to the main pathway of 1. Crystal structures and site-directed mutagenesis studies uncovered key residues for FAD-binding, substrate binding and catalytic activities, enabling the proposal for the CrmK catalytic mechanism. This study provides the first biochemical and structural evidence for flavoenzyme-mediated substrate recycling in secondary metabolism.

Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines.

Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts.

Peroxidase activity and involvement in the oxidative stress response of roseobacter denitrificans truncated hemoglobin.

Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.

Biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants.

Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core ß(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.

Human antibody response to N-glycans present on plant-made influenza virus-like particle (VLP) vaccines.

BACKGROUND: Plant-made biotherapeutics are gathering momentum and some plant glycoproteins are allergens. Glycans with core ß1-2xylose and α1,3fucose motifs and antennae terminated by mannose residues (e.g.: MMXF) are found on several plant allergens and can cross-react with glyco-epitopes from other sources. To date, reactivity to these cross-reactive determinants has not been associated with clinical symptoms. OBJECTIVE: We produced VLP vaccines bearing the hemagglutinin(HA) of H5(A/Indonesia/5/05) or H1(A/California/07/09) influenza viruses by transfection of Nicotiana benthamiana. Subjects enrolled in Phase I/II trials were followed for evidence of allergy/hypersensitivity and development of antibodies against plant glyco-epitopes. METHODS: A total of 280/349 subjects received either one (H1) or 2 doses (H5) of vaccine (5-45 µg of HA/dose) intramuscularly including 40 with pre-existing plant allergies. Subjects were monitored for 6 months. IgG and IgE to plant glyco-epitopes were measured by ELISA using corn-/egg-derived avidin and bromelain as target antigens. RESULTS: No subject developed allergic/hypersensitivity symptoms. Some (34%) developed transient IgG and, in some cases IgE, to plant glyco-epitopes but no subject mounted an IgE response to the MMXF motif. Antibodies returned to baseline by 6 months in most subjects. CONCLUSION: VLP vaccines bearing influenza HA glycoproteins can elicit transient IgG and, in some cases, IgE responses that are not associated with either the development or worsening of allergic/hypersensitivity symptoms.