Time filter

Source Type

Yusibov V.,Fraunhofer United States Center for Molecular Biotechnology | Kushnir N.,Fraunhofer United States Center for Molecular Biotechnology | Streatfield S.J.,Fraunhofer United States Center for Molecular Biotechnology
Annual Review of Plant Biology | Year: 2016

Monoclonal antibodies (mAbs) have a wide range of modern applications, including research, diagnostic, therapeutic, and industrial uses. Market demand for mAbs is high and continues to grow. Although mammalian systems, which currently dominate the biomanufacturing industry, produce effective and safe recombinant mAbs, they have a limited manufacturing capacity and high costs. Bacteria, yeast, and insect cell systems are highly scalable and cost effective but vary in their ability to produce appropriate posttranslationally modified mAbs. Plants and green algae are emerging as promising production platforms because of their time and cost efficiencies, scalability, lack of mammalian pathogens, and eukaryotic posttranslational protein modification machinery. So far, plant- and algae-derived mAbs have been produced predominantly as candidate therapeutics for infectious diseases and cancer. These candidates have been extensively evaluated in animal models, and some have shown efficacy in clinical trials. Here, we review ongoing efforts to advance the production of mAbs in plants and algae. Copyright © 2016 by Annual Reviews. All rights reserved.


Barry S.M.,University of Warwick | Barry S.M.,King's College London | Kers J.A.,Cornell University | Kers J.A.,Intrexon Corporation | And 12 more authors.
Nature Chemical Biology | Year: 2012

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N′-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O 2. © 2012 Nature America, Inc. All rights reserved.


Cummings J.F.,U.S. Army | Guerrero M.L.,U.S. Army | Moon J.E.,U.S. Army | Waterman P.,U.S. Army | And 6 more authors.
Vaccine | Year: 2014

Background: Novel influenza viruses continue to pose a potential pandemic threat worldwide. In recent years, plants have been used to produce recombinant proteins, including subunit vaccines. A subunit influenza vaccine, HAC1, based on recombinant hemagglutinin from the 2009 pandemic A/California/04/2009 (H1N1) strain of influenza virus, has been manufactured using a plant virus-based transient expression technology in Nicotiana benthamiana plants and demonstrated to be immunogenic and safe in pre-clinical studies (Shoji et al., 2011). Methods: A first-in-human, Phase 1, single-center, randomized, placebo-controlled, single-blind, dose escalation study was conducted to investigate safety, reactogenicity and immunogenicity of an HAC1 formulation at three escalating dose levels (15μg, 45μg and 90μg) with and without Alhydrogel®, in healthy adults 18-50 years of age (inclusive). Eighty participants were randomized into six study vaccine groups, a saline placebo group and an approved monovalent H1N1 vaccine group. Recipients received two doses of vaccine or placebo (except for the monovalent H1N1 vaccine cohort, which received a single dose of vaccine, later followed by a dose of placebo). Results: The experimental vaccine was safe and well tolerated, and comparable to placebo and the approved monovalent H1N1 vaccine. Pain and tenderness at the injection site were the only local solicited reactions reported following vaccinations. Nearly all adverse events were mild to moderate in severity. The HAC1 vaccine was also immunogenic, with the highest seroconversion rates, based on serum hemagglutination-inhibition and virus microneutralization antibody titers, in the 90. μg non-adjuvanted HAC1 vaccine group after the second vaccine dose (78% and 100%, respectively). Conclusions: This is the first study demonstrating the safety and immunogenicity of a plant-produced subunit H1N1 influenza vaccine in healthy adults. The results support further clinical investigation of the HAC1 vaccine as well as demonstrate the feasibility of the plant-based technology for vaccine antigen production. © 2014.


Kushnir N.,Fraunhofer United States Center for Molecular Biotechnology | Streatfield S.J.,Fraunhofer United States Center for Molecular Biotechnology | Yusibov V.,Fraunhofer United States Center for Molecular Biotechnology
Vaccine | Year: 2012

Virus-like particles (VLPs) are a class of subunit vaccines that differentiate themselves from soluble recombinant antigens by stronger protective immunogenicity associated with the VLP structure. Like parental viruses, VLPs can be either non-enveloped or enveloped, and they can form following expression of one or several viral structural proteins in a recombinant heterologous system. Depending on the complexity of the VLP, it can be produced in either a prokaryotic or eukaryotic expression system using target-encoding recombinant vectors, or in some cases can be assembled in cell-free conditions. To date, a wide variety of VLP-based candidate vaccines targeting various viral, bacterial, parasitic and fungal pathogens, as well as non-infectious diseases, have been produced in different expression systems. Some VLPs have entered clinical development and a few have been licensed and commercialized. This article reviews VLP-based vaccines produced in different systems, their immunogenicity in animal models and their status in clinical development. © 2012 Elsevier Ltd.


Madhun A.S.,University of Bergen | Haaheim L.R.,Myrdalskogen 95 | Nostbakken J.K.,University of Bergen | Ebensen T.,Helmholtz Center for Infection Research | And 4 more authors.
Vaccine | Year: 2011

Vaccination is the best available measure of limiting the impact of the next influenza pandemic. Ideally, a candidate pandemic influenza vaccine should be easy to administer and should elicit strong mucosal and systemic immune responses. Production of influenza subunit antigen in transient plant expression systems is an alternative to overcome the bottleneck in vaccine supply during influenza pandemic. Furthermore, a needle-free intranasal influenza vaccine is an attractive approach, which may provide immunity at the portal of virus entry. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with plant-derived influenza H5N1 (A/Anhui/1/05) antigen alone or formulated with bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) as adjuvant. The use of c-di-GMP as intramuscular adjuvant did not enhance the immune response to plant-derived influenza H5 antigen. However, intranasal c-di-GMP-adjuvanted vaccine induced strong mucosal and systemic humoral immune responses. Additionally, the intranasal vaccine elicited a balanced Th1/Th2 profile and, most importantly, high frequencies of multifunctional Th1 CD4+ cells. Our results highlight that c-di-GMP is a promising mucosal adjuvant for pandemic influenza vaccine development. © 2011 Elsevier Ltd.


Mamedov T.,Fraunhofer United States Center for Molecular Biotechnology
Bioengineered | Year: 2013

At present, several eukaryotic expression systems including yeast, insect and mammalian cells and plants are used for the production of recombinant proteins. Proteins with potential N-glycosylation sites are efficiently glycosylated when expressed in these systems. However, the ability of the eukaryotic expression systems to glycosylate may be not desirable for some proteins. If target proteins that do not carry N-linked glycans in the native host contain potential N-linked glycosylation sites, they can be aberrantly glycosylated in the eukaryotic expression systems, thus, potentially impairing biological activity. Recently, we have developed a strategy of enzymatic deglycosylation of proteins in vivo by co-introducing bacterial PNGase F via agroinfiltration followed by transient expression in plants. (1) Here, we summarize our work on this topic and its potential implications.


Buyel J.F.,Fraunhofer United States Center for Molecular Biotechnology | Buyel J.F.,RWTH Aachen | Bautista J.A.,Fraunhofer United States Center for Molecular Biotechnology | Fischer R.,RWTH Aachen | And 2 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model [18]. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1. g of protein per 1. kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50. g to 1. kg of processed leaf biomass. © 2011 Elsevier B.V.


Kropat J.,University of California at Los Angeles | Gallaher S.D.,University of California at Los Angeles | Urzica E.I.,University of California at Los Angeles | Urzica E.I.,Westfaelische Wilhelms University | And 6 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retentionof genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.


Wu Y.,National Institute of Allergy and Infectious Diseases | Sinden R.E.,The Jenner Institute | Churcher T.S.,Imperial College London | Tsuboi T.,Ehime University | Yusibov V.,Fraunhofer United States Center for Molecular Biotechnology
Advances in Parasitology | Year: 2015

Despite decades of effort battling against malaria, the disease is still a major cause of morbidity and mortality. Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. In the 1950s, Huff et al. first demonstrated the induction of transmission-blocking immunity in chickens by repeated immunizations with Plasmodium gallinaceum-infected red blood cells. Since then, significant progress has been made in identification of parasite antigens responsible for transmission-blocking activity. Recombinant technologies accelerated evaluation of these antigens as vaccine candidates, and it is possible to induce effective transmission-blocking immunity in humans both by natural infection and now by immunization with recombinant vaccines. This chapter reviews the efforts to produce TBVs, summarizes the current status and advances and discusses the remaining challenges and approaches. © 2015 Elsevier Ltd.


Mamedov T.,Fraunhofer United States Center for Molecular Biotechnology | Yusibov V.,Fraunhofer United States Center for Molecular Biotechnology
FEBS Open Bio | Year: 2011

Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii, a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry analysis of N-linked oligosaccharides released from total extracts of Chlamydomonas reinhardtii and demonstrated that C. reinhardtii algae possess glycoproteins with mammalian-like sialylated N-linked oligosaccharides. These findings suggest that C. reinhardtii may be an attractive system for expression of target proteins. © 2011 Federation of European Biochemical Societies.

Loading Fraunhofer United States Center for Molecular Biotechnology collaborators
Loading Fraunhofer United States Center for Molecular Biotechnology collaborators