Folding of influenza virus hemagglutinin in insect cells is fast and efficient
Li, X. ; Oers, M.M. van; Vlak, J.M. ; Braakman, I. - \ 2015
Journal of Biotechnology 203 (2015). - ISSN 0168-1656 - p. 77 - 83.
disulfide bond formation - endoplasmic-reticulum - quality-control - membrane glycoprotein - expression system - escherichia-coli - calnexin - vaccine - ph - oligomerization
Folding of influenza virus hemagglutinin (HA) in the endoplasmic reticulum has been well defined inmammalian cells. In different mammalian cell lines the protein follows the same folding pathway withidentical folding intermediates, but folds with very different kinetics. To examine the effect of cellularcontext on HA folding and to test to which extent insect cells would support the HA folding process,we expressed HA in Sf9 insect cells. Strikingly, in this invertebrate system HA folded faster and moreefficiently, still via the same folding intermediates as in vertebrate cells. Our results suggest that insectcells provide a highly efficient and effective folding environment for influenza virus HA and the idealproduction platform for HA (emergency) vaccines.
Effective Chikungunya Virus-like Particle Vaccine Produced in Insect Cells
Metz, S.W.H. ; Gardner, J. ; Geertsema, C. ; Le, T.T. ; Goh, L. ; Vlak, J.M. ; Suhrbier, A. ; Pijlman, G.P. - \ 2013
PLoS Neglected Tropical Diseases 7 (2013)3. - ISSN 1935-2727
equine encephalitis-virus - envelope proteins - baculovirus vectors - inactivated vaccine - expression system - dna vaccines - immunogenicity - infection - opportunities - glycosylation
The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV) causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.
Nicotiana benthamiana as a Production Platform for Artemisinin Precursors
Herpen, T.W.J.M. van; Cankar, K. ; Nogueira, M. ; Bosch, H.J. ; Bouwmeester, H.J. ; Beekwilder, M.J. - \ 2010
PLoS ONE 5 (2010)12. - ISSN 1932-6203 - 11 p.
antimalarial-drug artemisinin - expression system - molecular-cloning - plants - biosynthesis - annua - reductase - tobacco - yield - acid
Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.
In vitro analysis of protection of the enzyme bile salt hydrolase against enteric conditions by whey protein-gum arabic microencapsulation
Lambert, J.M. ; Weinbreck, F. ; Kleerebezem, M. - \ 2008
Journal of Agricultural and Food Chemistry 56 (2008)18. - ISSN 0021-8561 - p. 8360 - 8364.
lactococcus-lactis - lactobacillus-plantarum - gastrointestinal-tract - release properties - expression system - alginate - delivery - chitosan
The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable.