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Day 1 | Day 2 | Speaker Bios
Baculovirus vectors are widely used as tools for expressing proteins, delivering genes into cells, and creating vaccines. Once viewed as an alternative, the baculovirus system has achieved recognition through extensive clinical testing and regulatory exposure. The speed of reaching protein expression and the inability to transmit mammalian disease makes baculovirus an attractive platform.
The Baculovirus Technology meeting brings together experts who are innovating applications and achieving clinical success. Along with seminal insights into biological properties, case studies from industry leaders will address breakthrough approaches to increasing protein expression levels and humanizing the N-glycosylation pathway. Come explore the intricacies and successes of Baculovirus Technology.
Wednesday, August 25
7:30am Registration and Morning Coffee
8:20 Chairperson’s Remarks
Donald Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming
8:25 Opening Keynote Presentation:
Strategies for Baculovirus Replicative Success: Viral Induction and Inhibition of Apoptosis
Paul Friesen, Ph.D., Professor, Biochemistry & Institute for Molecular Virology, University of Wisconsin-Madison
The extraordinary productivity of baculovirus vectors for the expression of foreign genes in invertebrates is largely due to the efficiency with which these clever viruses counteract host anti-virus mechanisms and thereby achieve their enormous biosynthetic capabilities. Insects use diverse defense mechanisms for shutting down or limiting multiplication of DNA and RNA viruses. Of these, apoptosis is one of the most effective strategies whereby host cell suicide eliminates the pathogen and restricts virus dissemination within the animal. It is now clear that the replication potential of baculoviruses is greatly enhanced by their ability to block virus-induced apoptosis. For example, yields of AcMNPV drop >1,000 fold in apoptotic cells. Since the original discovery by Lois Miller’s group that baculoviruses encode potent suppressors of apoptosis, the inhibitor-of-apoptosis (IAP) proteins have provided the most insight into the apoptotic pathways of animals. First identified in baculoviruses, the IAPs are now recognized as critical regulators of life and death decisions in animals, insects included. These low abundance proteins control apoptosis by their ability to bind and neutralize caspases, the proteases responsible for the execution of apoptosis. We have recently discovered that host cell IAPs are rapidly depleted during infection of diverse insects and that this loss of IAP coincides with the activation of apoptotic caspases. When the endogenous pool of IAP was artificially elevated in these cells, virus-induced apoptosis was prevented or delayed. By using RNA silencing approaches, we demonstrated that baculovirus DNA replication triggers the depletion of endogenous IAP. Thus, the loss of intracellular IAP and the subsequent apoptotic response is an active process. Molecular mapping of protein domains within insect IAPs indicated that the first 30 residues at the IAP N terminus are sufficient to regulate this host-mediated turnover. Remarkably, baculovirus-encoded IAPs lack these residues and as a result are highly stable during infection. Our studies suggest that host insect IAPs function as critical sensors of virus infection that respond to virus-specific signals by rapid depletion and activation of apoptosis which destroys the invading virus. However, by expressing modified forms of cellular IAPs that are resistant to host-activated down regulation, the baculoviruses achieve complete inhibition of premature cell death and restore virus replication. These findings have important implications for strategies to improve baculovirus vector efficiency in both vertebrate and invertebrate hosts through the use of engineered IAPs for improved suppression of host-specific apoptosis. Moreover, these studies should provide a better understanding of the mechanisms by which insects control virus multiplication and how such control impacts the competency of insect hosts to vector human pathogenic viruses. Biography
9:00 Baculovirus and Generated Proteins for Structure Based Drug Design – More Lessons Learned
James Groarke, Ph.D., Fellow, DT Protein Structure, Novartis Institutes for BioMedical Research, Inc.
Generation of recombinant proteins in heterologous systems (specifically E.coli and Insect Cells) has become the workhorse for supplying proteins to support structure-based drug design. While these systems are generally reliable, many obstacles still remain in utilizing these expression systems to their full potential (expression levels, insolubility, scale-up issues, and post-translational modifications, for example). This talk will focus on recent advances in techniques and technologies that are being employed to overcome these aforementioned obstacles. Case Studies will be presented from the baculovirus/insect cell expression systems that highlight utilization of these technologies and improvements in the throughput process. Biography
9:30 Insights into Baculovirus Entry and a New Highly Productive Insect Cell Line
Gary Blissard, Ph.D., Scientist, Boyce Thompson Institute; Adjunct Professor, Department of Microbiology and Immunology and Department of Entomology, Cornell University
Infection by baculovirus budded virions in cell culture is mediated by the viral envelope glycoprotein, GP64. In addition to its role in entry, GP64 is also necessary for efficient budding of progeny virions. Recent studies in my lab have focused on the mechanism of GP64-mediated viral entry, including steps of virus attachment and membrane fusion. After virus attachment at the cell surface and endocytosis, the low pH of the endosome triggers a conformational change in GP64. That conformational change drives the fusion of adjacent viral and cellular membranes. Using site directed mutagenesis of GP64 in combination with a variety of functional assays, we have addressed both structural and functional questions associated with GP64 receptor binding, membrane fusion, and virion budding. We have also recently developed a new highly productive insect cell line named Ao38, from eggs of the Black Witch Moth (Ascalapha odorata). Ao38 cells are highly permissive for AcMNPV replication and Ao38 cells produced intracellular and secreted proteins at levels exceeding those of Sf9 and High Five cell lines. Biography
10:00 Networking Coffee Break with Exhibit and Poster Viewing
10:45 High-Throughput Methodology for the Rapid Screening of Gene Targets in a Baculovirus Platform
Jared Cartwright, Ph.D., Head, Protein Production Laboratory, Biology, University of York
We have developed a new set of tools for the parallel assembly of gene targets in a baculovirus platform with the subsequent rapid optimization of protein expression suitable for scale-up using bioreactor technology. The seminar will discuss all elements of baculovirus production including a rapid ligation-independent gene cloning platform suitable for the production of transfer vectors capable of intracellular and secreted protein expression. The vectors contain a copy of the green fluorescent protein (GFP) under the control of a separate promoter which serves as a marker for successful co-transfection and provides a simple mechanism to confirm viral infection. The GFP system has been further developed to provide a very simple, cheap and effective means of determining a viral titer estimation and has the advantage that it only reflects functional recombinant virus particles. We have further developed a rapid screening process to assess protein expression in suspension adapted micro-bioreactors (Applikon) and demonstrate that the parameters identified in the micro-bioreactors are readily scaled to production capacity in single-use bioreactors (scale circa 10 L). We believe that the integration of new and existing technology has produced a truly high-throughput baculovirus expression platform. Finally, we will demonstrate successful affinity purification of targets from both intracellular and secreted expression. Biography
11:15 Screening Methods to Efficiently Produce Functional Membrane Proteins for Structural Studies using Baculovirus Expression
Ted Fox, Ph.D., Senior Director, Biology, Vertex Pharmaceuticals, Inc.
Baculovirus production has become cheaper and easier, and is readily accessible to most laboratories. For this reason the number of deposited structures of BVES-derived proteins in the Protein Data Bank continues to accelerate each year. Membrane proteins present numerous expression challenges and again, BVES continues to expand its contribution in this area. In our experience, producing membrane proteins is usually not the critical issue. However, ensuring that the protein is correctly folded and functional is essential to success in biophysical and structural efforts. Some of the most useful and robust expression and biochemical screening methods we use to generate functional membrane proteins for biochemical, biophysical and structural studies will be described.
Sponsored by11:45 Sponsored Presentation I
Enhancements to Baculovirus Expression TechnologyNorm Garceau, Ph.D., CSO and President, Blue Sky Biotech
12:00pm Sponsored Presentation II (Opportunity Available)
12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own
1:55 Chairperson’s Remarks
Indresh Srivastava, Ph.D., Associate Director, Vaccines Research, Protein Biochemistry, Novartis Vaccines & Diagnostics, Inc.
2:00 Baculovirus Expression: A Tool for Producing Immunogens for In-Vivo Evaluation
2:30 Using a Novel Glyco-Engineered Insect Cell Line to Examine the Role of Glycan Structure in Influenza Virus Subunit Vaccine Efficacy
We created and characterized a transgenic insect cell line capable of producing recombinant influenza hemagglutinins with more authentic N-glycans. We produced rHA in these transgenic insect cells and in the native, parental insect cell line. We then compared the relative efficacy of these two forms of rHA, with mammalian-like or insect-like N-glycans, in an aerosol challenge assay in mice with an H5N1 influenza virus. The results indicated the rHA with mammalian N-glycans was more efficacious at a lower dose than the rHA with insect N-glycans. Biography
3:00 Utilizing the BEVS to Explore the Natural Immunity of Mosquitoes to Malaria
Richard Baxter, Ph.D., Assistant Professor, Department of Chemistry, Yale University
Malaria is the world's most devastating parasitic diseases. The search for novel malaria control strategies has led to investigation of the innate immune response of the vector Anopheles gambiae to infection by malarial parasites (genus Plasmodium). In particular, the complement-like protein thioester-containing protein 1 (TEP1) is a major factor in the destuction of Plasmodium ookinetes. I shall describe utilization of the BEVS in addressing structural and functional questions on the mechanism of TEP1-dependent killing of Plasmodium. Biography
3:30 Networking Refreshment Break with Exhibit and
4:15 Production of a Fast-Track Influenza Vaccine using Insect Cells
Penny Post, Ph.D., VP, Regulatory & Quality, Protein Sciences Corporation
Approximately 5–20% of the US population is infected with influenza annually, where > 200,000 are hospitalized from complications and 36,000 die from flu each year. Influenza is the 6th leading cause of death among US adults. All influenza vaccines currently licensed in the US are made in embryonated eggs. Protein Sciences Corporation uses the baculovirus expression vector system (BEVS) and insect cells to produce FluBlok, a novel influenza vaccine. This production system offers a rapid response advantage for the manufacturing of an influenza vaccine, with its annual changing composition. Other advantages include rapid response to emerging strains, elimination of the need to handle live viruses (especially for pandemic influenza), and production of authentic antigen that does not require egg-adaptation. FluBlok was granted Fast Track product status by the FDA and Protein Sciences Corporation anticipates licensure of FluBlok within the 2010/2011 influenza season. This talk will describe the technology used to produce this vaccine, the clinical trial data obtained, and regulatory considerations, including our path to product licensure. Biography
4:45 From Viral Vectors to VLPs: How the Production of AAV Vectors is Being Used as a Model
Marc Aucoin, Ph.D., Assistant Professor, Chemical Engineering, University of Waterloo
Over the past 8 years we have studied the production of adeno-associated viral vectors in insect cells following the co-infection of cells with three or more baculovirus vectors. This talk will highlight what we have learned and developed that can be applicable to the advancement of the baculovirus expression vector system for the production of VLPs and other molecules which require co-infection, including our success with low MOI high-density cultures and quantification of baculoviruses.
5:15 Reception in the Exhibit Hall (Sponsorship Available)
6:30 End of Day One
Day 1 | Day 2 | Speaker Bios
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