Increasing Complexity of the N-Glycome During Caenorhabditis Development.

Caenorhabditis elegans is a frequently employed genetic model organism and has been the object of a wide range of developmental, genetic, proteomic, and glycomic studies. Here, using an off-line MALDI-TOF-MS approach, we have analyzed the N-glycans of mixed embryos and liquid- or plate-grown L4 larvae. Of the over 200 different annotatable N-glycan structures, variations between the stages as well as the mode of cultivation were observed. While the embryonal N-glycome appears less complicated overall, the liquid- and plate-grown larvae differ especially in terms of methylation of bisecting fucose, α-galactosylation of mannose, and di-ß-galactosylation of core α1,6-fucose. Furthermore, we analyzed the O-glycans by LC-electrospray ionization-MS following ß-elimination; especially the embryonal O-glycomes included a set of phosphorylcholine-modified structures, previously not shown to exist in nematodes. However, the set of glycan structures cannot be clearly correlated with levels of glycosyltransferase transcripts in developmental RNA-Seq datasets, but there is an indication for coordinated expression of clusters of potential glycosylation-relevant genes. Thus, there are still questions to be answered in terms of how and why a simple nematode synthesizes such a diverse glycome.

A scalable, integrated downstream process for production of a recombinant measles virus-vectored vaccine.

Purification of very large and complex, enveloped viruses, such as measles virus is very challenging, it must be performed in a closed system because the final product cannot be sterile filtered and often loss of virus titer and poor product purity has been observed. We developed a purification process where the clarified and endonuclease treated culture supernatant is loaded on a restricted access chromatography medium where small impurities are bound and the virus is collected in the flow-through, which is then concentrated, and buffer exchanged by ultra/diafiltration. Up to 98.5% of host cell proteins could be captured by direct loading of clarified and endonuclease treated cell culture supernatant. Reproducible process performance and scalability of the chromatography step were demonstrated from small to pilot scale, including loading volumes from 50 mL up to 9 L. A 10-fold virus concentration was achieved by the ultrafiltration using a 100 kDa flat-sheet membrane. The order of individual process steps had a large impact on the virus infectivity and total process yields. The developed process maintained virus infectivity and is twice as fast as the traditional process train, where concentration is performed before loading on the chromatography column. Capturing impurities by the restricted access medium makes it a platform purification process with a high flexibility, which can be easily and quickly adapted to other vectors based on the measles virus vector platform.

High shear resistance of insect cells: the basis for substantial improvements in cell culture process design.

Multicellular organisms cultivated in continuous stirred tank reactors (CSTRs) are more sensitive to environmental conditions in the suspension culture than microbial cells. The hypothesis, that stirring induced shear stress is the main problem, persists, although it has been shown that these cells are not so sensitive to shear. As these results are largely based on Chinese Hamster Ovary (CHO) cell experiments the question remains if similar behavior is valid for insect cells with a higher specific oxygen demand. The requirement of higher oxygen transfer rates is associated with higher shear forces in the process. Consequently, we focused on the shear resistance of insect cells, using CHO cells as reference system. We applied a microfluidic device that allowed defined variations in shear rates. Both cell lines displayed high resistance to shear rates up to 8.73 × 105 s-1. Based on these results we used microbial CSTRs, operated at high revolution speeds and low aeration rates and found no negative impact on cell viability. Further, this cultivation approach led to substantially reduced gas flow rates, gas bubble and foam formation, while addition of pure oxygen was no longer necessary. Therefore, this study contributes to the development of more robust insect cell culture processes.

Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells.

Recombinant protein and virus-like particle (VLP) production based on the baculovirus expression vector system is fast, flexible, and offers high yields. Independent from the product, a multitude of parameters are screened during process development/optimisation. Early development acceleration is a key requirement for economic efficiency, and µ-scale bioreactor systems represent an attractive solution for high-throughput (HTP) experimentation. However, limited practical knowledge is available on the relevance and transferability of screening data to pilot scales and manufacturing. The main goal of the present study was to evaluate a HTP µ-bioreactor platform with respect to its aptitude as a screening platform mainly based on transferability of results to benchtop bioreactors representing the conventional production regime. Second question was to investigate to what extent the online sensors of the µ-bioreactor contribute to process understanding and development. We demonstrated that transferability of infection screening results from the HTP µ-bioreactor scale to the benchtop bioreactor was equal or better than that from shaker cultivation. However, both experimental setups turned out to be sub-optimal solutions that only allowed for a first and rough ranking with low relevance in the case of absolute numbers. Bioreactor yields were up to one order of magnitude higher than the results of screening experiments.

Development of a novel Ara h 2 hypoallergen with no IgE binding or anaphylactogenic activity.

BACKGROUND: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects. OBJECTIVE: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity. METHODS: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract. RESULTS: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice. CONCLUSIONS: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated.

Development of a Dual-Vector System Utilizing MicroRNA Mimics of the Autographa californica miR-1 for an Inducible Knockdown in Insect Cells.

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years, several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study, an inducible knockdown system was established in insect (Sf9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next, recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting Sf9 cells with these two viruses, the fluorescence intensity of eYFP was suppressed by ~30⁻40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform.

Expression of full-length HER2 protein in Sf9 insect cells and its presentation on the surface of budded virus-like particles.

Biomarkers of cancer are often glycosylated membrane receptor proteins present on the cellular surface. In order to develop new antibodies for cancer diagnostics or treatment, it is a main pre-requisite that these target proteins are available in a native conformation. However, membrane receptor proteins are notoriously difficult to produce due to their hydrophobic nature and complex architecture. Here, we used the baculovirus-insect cell expression system to produce budded virus-like particles (VLPs) as the scaffold for the presentation of complex membrane proteins. Since the human epidermal growth factor receptor 2 (HER2) is known to be overexpressed in a number of cancers it was chosen as model for a tumor antigen. VLPs displaying full-length HER2 on the surface were produced in Spodoptera frugiperda 9 (Sf9) insect cells and purified by sucrose gradient ultracentrifugation. The number of secreted particles was quantified by nanoparticle tracking analysis. To confirm the presence of HER2 protein on the surface, VLPs were labeled with gold-conjugated antibodies and analyzed by transmission electron microscopy. Functionality of displayed HER2 was investigated by ELISA and a newly established biolayer interferometry based technique. Detection was accomplished using the specific monoclonal antibody Herceptin and filamentous phages displaying a single-chain variable fragment of an anti-HER2 antibody. Significant stronger binding of Herceptin and anti-HER2 phages to HER2-displaying VLPs as compared to control VLPs was demonstrated. Thus, we suggest that Sf9 insect cells are highly feasible for the fast and easy production of various budded VLPs that serve as a platform for full-length membrane receptor presentation.

The underestimated N-glycomes of lepidopteran species.

BACKGROUND: Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g., in case of pathogen binding or production of humanised antibodies; for a long time, it has been considered that insect N-glycosylation potentials are rather similar and simple, but as more species are glycomically analysed in depth, it is becoming obvious that there is indeed a large structural diversity and interspecies variability. METHODS: Using an off-line LC-MALDI-TOF MS approach, we have analysed the N-glycomes of two lepidopteran species (the cabbage looper Trichoplusia ni and the gypsy moth Lymantria dispar) as well as of the commonly-used T. ni High Five cell line. RESULTS: We detected not only sulphated, glucuronylated, core difucosylated and Lewis-like antennal fucosylated structures, but also the zwitterion phosphorylcholine on antennal GlcNAc residues, a modification otherwise familiar from nematodes; in L. dispar, N-glycans with glycolipid-like antennae containing α-linked N-acetylgalactosamine were also revealed. CONCLUSION: The lepidopteran glycomes analysed not only display core α1,3-fucosylation, which is foreign to mammals, but also up to 5% anionic and/or zwitterionic glycans previously not found in these species. SIGNIFICANCE: The occurrence of anionic and zwitterionic glycans in the Lepidoptera data is not only of glycoanalytical and evolutionary interest, but is of biotechnological relevance as lepidopteran cell lines are potential factories for recombinant glycoprotein production.

Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

SweetBac: Applying MultiBac Technology Towards Flexible Modification of Insect Cell Glycosylation.

Observed different glycosylation patterns of glycoconjugates (recombinantly) produced in various eukaryotic organisms are a direct consequence of differences in numerous proteins involved in biosynthesis of the relevant glycan chains in these species. The need for efficient, robust and flexible methods for recombinant expression of proteins is met by the recently described MultiBac technology, an advanced and optimized baculovirus-based system for simultaneous recombinant protein expression in insect cells. A derivative of MultiBac technology, the SweetBac system aims at the modification of the glycosylation potential of insect cells as expression hosts. The application of SweetBac, including the methods needed to investigate the glycosylation pattern of the purified recombinant protein, is described in this chapter.

Atopic donor status does not influence the uptake of the major grass pollen allergen, Phl p 5, by dendritic cells.

Dendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming. In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1). In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes. The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays.

Minimizing fucosylation in insect cell-derived glycoproteins reduces binding to IgE antibodies from the sera of patients with allergy.

The baculovirus/insect cell system has proven to be a very powerful tool for the expression of several therapeutics. Nevertheless, these products sometimes suffer from reduced biological activity and unwanted side effects. Several studies have demonstrated that glycosylation can greatly influence the structure, function, half-life, antigenicity and immunogenicity of various glycoproteins. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Especially, the presence of core α1,3-linked fucose bears the risk of causing immediate hypersensitivity reactions in patients with allergy. In this study, we evaluated the impact of fucose residues on the allergenic potential of an insect cell-expressed vaccine candidate. In order to block the GDP-L-fucose de novo synthesis pathway, we integrated the Pseudomonas aeruginosa GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene into a baculovirus backbone. This virus was then used for the expression of soluble influenza A virus hemagglutinin (HA). Expression studies showed that the co-expression of RMD did not influence the overall level of recombinant protein secretion. We confirmed the result of our strategy by analyzing PNGase A-released N-glycans using MALDI-TOF-MS. In order to evaluate the biological impact of defucosylation of influenza HA we tested the binding activity of IgE derived from the sera of patients with allergy to the purified antigen. The non-fucosylated HA showed a 10-fold decrease in IgE binding levels as compared to wildtype variants.

One-shot vaccination with an insect cell-derived low-dose influenza A H7 virus-like particle preparation protects mice against H7N9 challenge.

Human infections with a novel influenza A H7N9 subtype virus were reported in China recently. The virus caused severe disease with high mortality rates and it raised concerns over its pandemic potential. Here, we assessed in the mouse model protective efficacy of single immunisations with low vaccine doses of insect cell-derived H7 virus-like particles, consisting of hemagglutinin and matrix protein. Vaccinated mice were fully protected and survived a stringent lethal challenge (100 mLD50) with H7N9, even after a single, unadjuvanted, low vaccine dose (0.03 µg). Serum analysis revealed broad reactivity and hemagglutination inhibition activity across a panel of divergent H7 strains. Moreover, we detected significant levels of cross-reactivity to related group 2 hemagglutinins. These data demonstrate that virus-like particle vaccines have the potential to induce broadly protective immunity against the novel H7N9 virus and a variety of other H7 strains.

Tnao38, high five and Sf9--evaluation of host-virus interactions in three different insect cell lines: baculovirus production and recombinant protein expression.

PURPOSE OF WORK: A comparative analysis of new and established insect cell lines, in regard to process relevant parameters, provide data that can be exploited for designing more robust and effective protein production processes. The baculovirus-insect cell expression system has been efficiently used for the production of heterologous proteins. Three different insect cell lines Tnao38, High Five and Sf9 were compared in terms of virus susceptibility, baculovirus production and product yield of an intra-cellularly (YFP) and extra-cellularly (influenza A virus hemagglutinin)-expressed recombinant protein. The Tnao38 and High Five cell lines exhibited higher (tenfold) susceptibility to baculovirus infection than Sf9 cells, whereas Sf9 cells showed a higher (100-fold) capacity for production of infectious virus particles. Analysis of recombinant protein expression revealed considerably higher product yields in Tnao38 and High Five cells as compared to Sf9 cells, for both model proteins. Overall, the two Trichoplusia ni-derived cell lines, High Five and Tnao38, were significantly more efficient in terms of secreting proteins such as the glycoprotein hemagglutinin of influenza A virus.

MultiBac turns sweet.

The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Therapeutics, especially in the field of vaccinology, are often composed of several different protein subunits. Conventional baculoviral expression schemes largely lack efficient strategies for simultaneous multi-gene expression. The MultiBac technology which is based on an engineered genome of Autographa californica nuclear polyhedrosis virus in combination with specially designed transfer vectors is an elegant way for flexible generation of multi-subunit proteins in insect cells. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Therefore, a modified version of MultiBac, SweetBac, was generated allowing for a flexible glycosylation of target proteins in insect cells. Beyond the SweetBac technology MultiBac can further be designed for bridging the gap between cell engineering and transient modulation of host genes for improved and product tailored expression of recombinant proteins.

MultiBac complexomics.

Recombinant production of multiprotein complexes is an emerging focus in academic and pharmaceutical research and is expected to play a key role in addressing complex biological questions in health and disease. Here we describe MultiBac, a state-of-the-art eukaryotic expression technology utilizing an engineered baculovirus to infect insect cells. The robust and flexible concept of MultiBac allows for simultaneous expression of multiple proteins in a single cell, which can be used to produce protein complexes and to recapitulate metabolic pathways. The MultiBac system has been set up as an open-access platform technology at the European Molecular Biology Laboratory (EMBL) in Grenoble, France. The performance of this platform and its access modalities to the scientific community are detailed in this article. The MultiBac system has been instrumental for unlocking the function of a number of essential multiprotein complexes and recent examples are discussed. This article presents a novel concept for the customized production of glycosylated protein targets using SweetBac, a modified MultiBac vector system. Finally, this article outlines how MultiBac may further develop in the future to serve applications in both academic and industrial research and development.

SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells.

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine ß1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.

Insect cells for antibody production: evaluation of an efficient alternative.

In recent years there has been an increase in both availability and demand for therapeutic monoclonal antibodies. Currently, most of these antibodies are produced by stably transfected mammalian cells. In this study we evaluated the use of different baculoviral insect cell systems as an alternative for commonly used production schemes. We expressed the human anti-gp41 antibody 3D6 in Spodoptera frugiperda Sf9, Trichoplusia ni BTI-TN5B1-4 "High Five", and Spodoptera frugiperda SfSWT-1 "Mimic™" insect cells and compared product yield, specificity and glycosylation patterns with a 3D6 antibody expressed in Chinese hamster ovary cells. Using "High Five" cells we achieved amounts of secreted antibody comparable to those resulting from transient expression in mammalian cells. We determined the N-linked oligosaccharide structures present on asparagine-297 in IgG1 heavy chains and tested the functionality in terms of antigen binding and the ability to elicit effector functions. Antibodies expressed in all insect cell lines displayed highly specific antigen binding. In general, the insect-produced antibodies carried, as the CHO-produced form, fucosylated N-glycans, including, in the case of "High Five" cells, high levels of core α1,3-fucose. This indicates that in all systems glycoengineering may be required in order to produce optimal glycoforms of this antibody.

Evaluation of the influenza A replicon for transient expression of recombinant proteins in mammalian cells.

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells.

Influenza virus-like particles as an antigen-carrier platform for the ESAT-6 epitope of Mycobacterium tuberculosis.

Various virus-like particles (VLPs) have been shown to induce cytotoxic T-cell immune response as well as B-cell immune response. This makes VLPs promising candidates for antigen-carrier platforms for various epitopes. Influenza A VLPs were produced displaying a 20 amino acid sequence from Mycobacterium tuberculosis early secretory antigenic target 6 protein (ESAT-6). As this sequence is known to comprise a potent T-cell epitope it was chosen as a model for a foreign epitope to be presented on an influenza VLP scaffold. The ESAT-6 epitope was engineered into the antigenic region B of the influenza hemagglutinin (HA) from strain A/New Caledonia/20/99. VLPs were expressed in insect cells and subjected to immunization studies in mice. High serum antibody titers detected against recombinant ESAT-6 demonstrated the feasibility of influenza A VLPs serving as an efficient platform for epitope presentation.