Inger Heldal
Senioringeniør
Forfattere
Ana-Maria Madalina Pantazica Andre van Eerde Mihaela-Olivia Dobrica Iuliana Caras Irina Ionescu Adriana Costache Catalin Tucureanu Hege Særvold Steen Catalin Lazar Inger Heldal Sissel Haugslien Adrian Onu Crina Stavaru Norica Branza-Nichita Jihong Liu ClarkeSammendrag
The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with “humanized” N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of β-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.
Forfattere
Nina Elisabeth Nagy Hans Ragnar Norli Monica Fongen Runa Berg Østby Inger Heldal Jahn Davik Ari HietalaSammendrag
Tree defense against xylem pathogens involves both constitutive and induced phenylpropanoids and terpenoids. The induced defenses include compartmentalization of compromised wood with a reaction zone (RZ) characterized by polyphenol deposition, whereas the role of terpenoids has remained poorly understood. To further elucidate the tree–pathogen interaction, we profiled spatial patterns in lignan (low-molecular-weight polyphenols) and terpenoid content in Norway spruce (Picea abies) trees showing heartwood colonization by the pathogenic white-rot fungus Heterobasidion parviporum. There was pronounced variation in the amount and composition of lignans between different xylem tissue zones of diseased and healthy trees. Intact RZ at basal stem regions, where colonization is the oldest, showed the highest level and diversity of these compounds. The antioxidant properties of lignans obviously hinder oxidative degradation of wood: RZ with lignans removed by extraction showed significantly higher mass loss than unextracted RZ when subjected to Fenton degradation. The reduced diversity and amount of lignans in pathogen-compromised RZ and decaying heartwood in comparison to intact RZ and healthy heartwood suggest that α-conindendrin isomer is an intermediate metabolite in lignan decomposition by H. parviporum. Diterpenes and diterpene alcohols constituted above 90% of the terpenes detected in sapwood of healthy and diseased trees. A significant finding was that traumatic resin canals, predominated by monoterpenes, were commonly associated with RZ. The findings clarify the roles and fate of lignan during wood decay and raise questions about the potential roles of terpenoids in signal transduction, synthesis, and translocation of defense compounds upon wood compartmentalization against decay fungi.
Forfattere
Hang Su Andre van Eerde Hege Særvold Steen Inger Heldal Sissel Haugslien Irene Ørpetveit Stefanie Caroline Wüstner Makoto Inami Marie Løvoll Espen Rimstad Jihong Liu ClarkeSammendrag
Cardiomyopathy syndrome (CMS) is a severe cardiac disease occurring in the grow-out sea phase of farmed Atlantic salmon with approximately 100 outbreaks annually in Norway. Piscine myocarditis virus (PMCV) is believed to be the causative agent of CMS. There is no vaccine available to control CMS, partially because PMCV withstands propagation in known cell cultures. In the present study, we selected the putative capsid protein of PMCV as the candidate antigen for immunization experiments and produced it in the plant Nicotiana benthamiana by transient expression. The recombinant PMCV antigen formed virus-like particles (VLPs). To evaluate the efficacy of the plant made VLP vaccine, a PMCV infection model was established. In an experimental salmon vaccination trial, the VLP vaccine triggered innate immunity, and indicative but not significant inhibition of viral replication in heart, spleen and kidney tissues was observed. Similarly, a reduction of inflammatory lesions in cardiomyocytes and subendocardial infiltration by mononuclear leukocytes were observed. Therefore, there was no difference in efficacy or immune response observed post the plant made PMCV VLP antigen vaccination. Taken together, this study has demonstrated that plant made VLP antigens should be investigated further as a possible platform for the development of PMCV antigens for a CMS vaccine.
Sammendrag
Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient factors of innate immunity, which are produced by all multicellular organisms and play a key role in their protection against infection. Red king crab (Paralithodes camtschaticus), also called Kamchatka crab, is widely distributed and the best known species of all king crabs belonging to the family Lithodidae. Despite their economic importance, the genetic resources of king crabs are scarcely known and no fullgenome sequences are available to date. Therefore, analysis of the red king crab transcriptome and identifcation and characterization of its AMPs could potentially contribute to the development of novel antimicrobial drug candidates when antibiotic resistance has become a global health threat. In this study, we sequenced the P. camtschaticus transcriptomes from carapace, tail fap and leg tissues using an Illumina NGS platform. Libraries were systematically analyzed for gene expression profles along with AMP prediction. By an in silico approach using public databases we defned 49 cDNAs encoding for AMP candidates belonging to diverse families and functional classes, including buforins, crustins, paralithocins, and ALFs (anti-lipopolysaccharide factors). We analyzed expression patterns of 27 AMP genes. The highest expression was found for Paralithocin 1 and Crustin 3, with more than 8,000 reads. Other paralithocins, ALFs, crustins and ubiquicidins were among medium expressed genes. This transcriptome data set and AMPs provide a solid baseline for further functional analysis in P. camtschaticus. Results from the current study contribute also to the future application of red king crab as a bio-resource in addition to its being a known seafood delicacy.
Forfattere
Andre van Eerde Aniko Varnai John-Kristian Jameson Lisa Paruch Anders Moen Jan Haug Anonsen Piotr Chylenski Hege Særvold Steen Inger Heldal Ralph Bock Vincent Eijsink Jihong Liu ClarkeSammendrag
Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.
Forfattere
Jihong Liu Clarke Andre van Eerde Lisa Paruch Inger Heldal Hege Særvold Steen Yanliang Wang Astrid Sivertsen Sissel HaugslienSammendrag
Det er ikke registrert sammendrag
Forfattere
Jihong Liu Clarke Lisa Paruch Hege Særvold Steen Andre van Eerde Yanliang Wang Astrid Sivertsen Sissel Haugslien Inger HeldalSammendrag
Det er ikke registrert sammendrag
Sammendrag
1. Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio-insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV. 2. In the present study, we developed real-time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real-time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real-time assay is 0.013 pg of viral DNA (0.013 × 10−12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample. 3. qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy- or bioassay-based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying. 4. This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).
Forfattere
Nina Elisabeth Nagy Carl Gunnar Fossdal Lars Sandved Dalen Anders Lønneborg Inger Heldal Øystein JohnsenSammendrag
Det er ikke registrert sammendrag
