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PROGRAM CHAIRS:
Penny L. Post, PhD - Protein Sciences Corporation
Monique M. van Oers, PhD - Wageningen University

Wian A. de Jongh, PhD
CSO, ExpreS2ion Biotechnologies
Development of Malaria Sub-Unit Vaccines Using the
Non-Viral Drosophila S2 Insect Cell Expression System

Abstract

Drosophila S2 insect cell expression is less known than the extensively used Spodoptera (Sf9/Sf21) or Trichoplusia ni (Hi-5) insect cell based baculovirus expression system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late stage Drosophila melanogaster (fruit fly) embryos by Schneider in the 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D.mel. 2). The S2 expression system has been used for antigen manufacture up to Phase II clinical trials, and was used, among others, for production of Dengue antigens tested in a Phase I trial by Merck Inc.

ExpreS2ion has developed S2-based production processes for two malaria vaccine clinical trails with The Jenner Institute, Oxford University (Rh5, blood-stage malaria) and Copenhagen University (VAR2CSA, pregnancy associated malaria). The placental malaria vaccine is currently in a Phase Ia trail in Germany, and will be followed by a Phase Ib trial in Benin. The blood-stage malaria vaccine has recently completed cGMP manufacture and the Phase I/IIa trial started in October 2016. Furthermore, transmission blocking malaria vaccine candidates (Pfs25, Pfs48/45, Pfs230C) are also under development. Antigen expression screening, process development, and GMP manufacture for these malaria programs will be discussed.

Biography

Dr. de Jongh (South African) obtained a bachelor's degree followed by an MSc in Chemical Engineering from the University of Stellenbosch, South Africa. Thereafter, he was awarded a doctorate in Biotechnology from the Technical University of Denmark (DTU, Denmark) in 2006.

During his PhD, Dr. de Jongh developed advanced cell line genetic engineering tools and applied metabolic engineering methodologies to cell lines with engineered improved production characteristics. Dr. de Jongh has significant experience in the pharmaceutical industry in molecular biology, cell line development, project management, upstream process development, process scale-up, and process transfer to cGMP manufacturing.

He was instrumental in the development of the proprietary expression vector system ExpreS2ion Biotechnologies was founded on. Dr. de Jongh has also served on the steering committee and as project manager on several grant-funded projects.

Luis A. Hernandez, PhD
Senior Scientist, Biological R&D – Molecular, Boehringer Ingelheim Vetmedica, Inc.
Baculovirus-Derived Particle and Subunit Vaccines for Influenza in Swine

Abstract

Influenza A virus (IAV) is an economically important pathogen involved in the porcine respiratory disease complex (PRDC), a major cause of mortality in swine. Although initially uncomplicated, the landscape of IAV strains in swine has developed considerable complexity over the last 20 years, making development of vaccines for control of IAV infection particularly challenging. As the goal of a universal influenza vaccine remains elusive, a successful IAV vaccine must elicit a broad immune response to contain the currently circulating IAV viruses while being amenable to rapid modification in the face of newly emerging strains. The baculovirus expression vector system has proven to be a flexible platform for the expression of recombinant forms of hemagglutinin. Subunit, virus-like particle (VLP)-displayed, and baculovirus-displayed hemagglutinin vaccines have been demonstrated to provide efficacy against influenza challenge in mice and other small animals. Unfortunately, little is known about their applicability in larger animals like swine. This presentation will detail the generation and evaluation of one subunit and two particle-based hemagglutinin vaccines and demonstrate their utility as a potential platform for IAV vaccine development for swine.

Biography

Luis Hernandez obtained his master’s degree at the University of Oklahoma Health Sciences Center prior to beginning a career in veterinary vaccine development with Intervet in 2002. Luis has spent the last 14 years working in both the early research and product development phases of the animal health industry with broad experience in construct design and optimization, antibody and assay development, and bioprocess design and scale-up. In 2015, Luis completed his doctoral degree in veterinary microbiology at Iowa State University while working at Boehringer Ingelheim Vetmedica, Inc. Today, Luis leads a small group focused on the development and evaluation of potential swine vaccine candidates utilizing baculovirus and E. coli-based platforms.

Nicola A. Burgess-Brown, PhD
Principal Investigator Biotechnology, The Structural Genomics Consortium
Optimising Baculovirus and BacMam Expression of Human Membrane Proteins

Abstract

Production of human integral membrane proteins (IMPs) for structural studies, although still challenging, has advanced significantly over the past couple of years. Since 2004, the Structural Genomics Consortium (SGC) globally has solved more than 1800 soluble human protein structures in addition to seven novel IMPs. These recombinant proteins have provided a rich resource for functional genomics, small molecule inhibitor screens, and generation of antibodies. Our established expression systems using baculovirus/insect cells will be presented as well as our BacMam process and optimisation strategies for improved mammalian expression screening.

Biography

Nicola Burgess-Brown obtained her degree in Applied Biochemical Sciences in 1997, then worked as a molecular biologist for SmithKline Beecham. She received her PhD in Molecular Microbiology at the University of Nottingham in 2001, then returned to industry to work on high-throughput cloning and validation of therapeutic cancer antigens for Oxford Glycosciences. Nicola is currently the Principal Investigator of the Biotech Group at the SGC, responsible for molecular biology, cell culture, protein production, and mass spectrometry analysis of the targets of interest at the Oxford site. Her group also develops methods for increasing protein expression and driving throughput.

Donald L. Jarvis, PhD
Professor, Molecular Biology, University of Wyoming
Recent Progress Towards Host Cell Improvement in the Baculovirus-Insect Cell System

Abstract

For the past 30 years, my group has undertaken basic and applied research efforts to improve baculovirus-insect cell technology and advance the baculovirus-insect cell system (BICS) as a recombinant protein production platform. One of our major efforts has focused on engineering protein glycosylation pathways to create new BICS capable of producing “humanized” recombinant glycoproteins. In this presentation, I will briefly overview our progress in this area and also report our new efforts to develop CRISPR-Cas9 tools for site-specific genome editing in the baculovirus-insect cell system. I will describe successful development and characterization of these tools and disclose some unexpected features we discovered along the way. I also will describe results obtained when we used these new tools for glycoengineering with an endogenous Spodoptera frugiperda (Sf) glycogene as the target. If time permits, this will be followed by a brief description of our efforts to use different glycoengineered BICS to produce recombinant proteins with various glycosylation patterns and study the impact of glycan structure on protein structure and/or function. Finally, this presentation will end with an overview and update of the properties of an Sf-rhabdovirus-negative Sf cell line created in our lab, which will include new results obtained by using this virus-free cell line to assess Sf-rhabdovirus infectivity, contamination, and to assess the possibility of contamination with other adventitious agents.

Biography

Don Jarvis earned a PhD in virology in 1986 at Baylor College of Medicine, where he studied biosynthesis and processing of the SV40 large tumor antigen. Don moved to Texas A&M University in 1987, where he joined Max Summers' group to initiate a research program focused on protein biosynthesis and processing in the BICS. Don was a faculty member at Texas A&M from 1989 to 1997, then moved his program to the University of Wyoming, where he continues to address protein processing and broader issues in an effort to improve baculovirus-insect cell technology and advance the BICS as a recombinant protein production platform. In 2011, Don started GlycoBac, LLC in collaboration with Christoph Geisler to take advantage of opportunities to fine-tune, commercialize, and apply new BICS and BICS components created in the academic lab.

João M.N. Vidigal
PhD Student, Instituto de Biologia Experimental e Tecnológica (iBET)
Insect Cell Platforms for Fast Production of Pseudotype Virus-Like Particles
for Drug and Vaccine Development

Abstract

Expression systems capable of delivering high concentrations of membrane proteins in their native structure are essential in the vaccine field as well as in drug discovery. In this work, we took advantage of insect cell expression and site-specific gene integration based on flipase-mediated cassette exchange (FMCE) technology to generate cell platforms for efficient production of membrane proteins on the surface of a protein scaffold, namely enveloped virus-like particles (VLPs). The expression of membrane proteins concomitantly with capsid proteins of enveloped viruses (e.g. HIV Gag or influenza M1) will enable their capturing in lipid rafts of the cellular plasma membrane and their display on the surface of budding VLPs, thus providing a native conformation for downstream assays.

Parental insect Sf-9 and High Five cells were randomly tagged with GFP-fused Gag or M1 proteins and FACS enriched with cells tagged in genomic “hot-spots” supporting high expression. A linker including a Flp recognition target (FRT) site was used to allow posterior removal of the marker gene from the particle through cassette exchange. By confocal microscopy we could observe that Gag localizes preferentially at the plasma membrane whereas M1 disperses within the cell. Upon promoting Flp-mediated recombination in the tagging populations, cassette exchange was well succeeded, allowing the recovery of cells tagged in loci supporting FMCE. We have evaluated the capability of both core proteins as scaffolds to display GPCRs (e.g. beta-2 adrenergic receptor) and influenza HA proteins. For the latter, we will present recent results on the feasibility of combining different bioprocess optimization strategies in order to improve cell line specific productivities. Overall, modular insect cells platforms are being generated to be readily adaptable for production of a broad range of VLP-based vaccines as well as receptor display particles for drug screening or antibody discovery.

Biography

João Vidigal is a last year PhD student in the field of biotechnology at the Animal Cell Technology Unit, iBET – Instituto de Biologia Experimental e Tecnológica and at Universidade NOVA de Lisboa. He has a degree in molecular biology and a Master's in cellular biology and biotechnology from Faculdade de Ciências da Universidade de Lisboa.

His main research interest focuses on stable insect cell line expression systems, mainly in bridging the gap between healthcare and vaccinology needs with improved production technologies targeting complex biologicals.

Brian Paszkiet
Staff Scientist, Thermo Fisher Scientific
A Yeastolate-Free Baculovirus-Based Expression System
for Enhanced Protein Production in Sf9 Cells

Abstract

The baculovirus expression vector system (BEVS) is one of the major protein expression platforms used for recombinant protein production. Unlike mammalian expression systems that have transitioned to serum-free, chemically-defined culture media for improved consistency and expression levels, this transition has not yet taken place with insect expression systems, with insect cells continuing to rely on undefined, yeastolate-containing culture media exhibiting significant lot-to-lot variability and limiting protein expression levels.

Here, we present the first data on the development of a novel Sf9-based baculovirus expression system based upon a yeastolate-free, animal origin-free, chemically-defined, high-density culture medium that allows for Sf9 cells to reach densities nearly twice as high as those attained in traditional yeastolate-containing media. Additionally, Sf9 cells adapted to grow to high densities in the yeastolate-free media were generated and a new, high-efficiency bacmid transfection reagent was developed to allow for the generation of high titer baculovirus stocks. Together, these improvements allow for the optimization of a new expression protocol that takes advantage of the high cell densities achievable with the new chemically-defined medium and adapted Sf9 cells, as well as high multiplicity of infection (MOI), to significantly improve protein titers and ensure lot-to-lot consistency of both cell growth and protein expression in a defined media formulation.

Biography

Brian has been working at Thermo Fisher Scientific since 2007, where his projects have focused on the development of cell culture products in the mammalian cell culture, insect cell culture, and protein expression fields. Previous to Thermo Fisher, Brian was in the cell therapy field, developing optimized media and viral reagents used for HIV as well as ocular disease therapy. This was where his use of transient transfection began, as a means to produce lentivirus vectors. His current focus is on the development of improved products for protein expression in insect cells.

Monique M. van Oers, PhD
Professor, Laboratory of Virology, Wageningen University
Per Os Infectivity Factors (PIF) are Essential Proteins of
Occlusion-Derived Viruses (ODVs), the ‘Other’ Baculovirus Particles

Abstract

Baculoviruses infect insect midgut epithelial cells via occlusion-derived virions (ODVs). ODVs are assembled in the nucleus of infected cells and are packaged in virus-encoded occlusion bodies (OBs). A set of eight conserved ODV envelope proteins is essential for oral infectivity of the insect’s midgut; hence, they are called per os infectivity factors (PIFs). In the ODV envelope a multi-molecular complex composed of PIF1, PIF2, PIF3, and PIF4 was found. Recently, using co-IP and non-denaturing gel electrophoresis, we showed that PIF6 (Ac68) is also present in the PIF complex. P74 (PIF0) is more loosely associated with this complex. Using the same techniques PIF5 was not found to associate with this complex and therefore, may have a separate function in oral infectivity. The same may hold true for PIF7 (Ac110). Co-IP experiments also indicated that P95 (Ac83, PIF8) interacts with the PIF-complex. P95 was recently described as a dualistic protein, with a domain required for nucleocapsid assembly and a second region, with typical PIF characteristics, crucial for PIF complex assembly. Gene deletions of either PIF1, PIF2, or PIF3 also abolished complex formation. When PIF4 or PIF6 were deleted, the PIF complex became more susceptible to degradation by larval proteases, co-purified with the OBs. To visualize the entry of ODVs into primary midgut epithelium cells and to analyze the role of individual PIF proteins therein, we constructed a baculovirus with a fluorescently labeled nucleocapsid, by fusing EGFP to the major capsid protein VP39. So far, we applied this technique to wild-type AcMNPV and viruses with deletions of P74, PIF1, or PIF2. In ex vivo binding and fusion assays with midgut epithelial cells, using live-imaging microscopy, we were able to show that P74 is absolutely required for ODV binding. Viruses with PIF1 or PIF2 deletions that were impaired in PIF-complex formation were still able to bind midgut epithelial cells, but did not enter these cells, suggesting that fusion was somehow impaired. The PIF-complex is likely to mediate an ancient and well-conserved entry mechanism of large nuclear invertebrate DNA viruses, since PIF proteins are also encoded by viruses in the families Nudiviridae, Hytrosaviridae, and Nimaviridae.

Biography

Professor Dr. Monique van Oers (PhD in 1994) is chair holder of the Laboratory of Virology at Wageningen University in the Netherlands. The laboratory focuses on insect viruses, plant viruses, and arboviruses. Her main scientific interest lies in fundamental and applied aspects of insect viruses and how viruses in general interact with insects, as host or vector. Current topics include functional genomics and evolution of large insect DNA viruses, and the molecular mechanisms behind baculovirus entry and packaging. An intriguing new research topic is the molecular mechanism behind virus-induced changes in insect behaviour. She has long-term experience in the exploitation of fundamental data to optimize the baculovirus insect-cell expression system for the production of recombinant proteins, i.e. vaccines and diagnostics, and for the production of gene therapy vectors. Her current interest here is to generate arbovirus vaccines and to develop a Bac-Free system that uses baculovirus technology to produce recombinant proteins without contaminating baculovirus particles. Several of the PhD students trained in her laboratory are/were involved in baculovirus-based vaccines against emerging human and fish diseases. She is trustee of the Society for Invertebrate Pathology. She has published over 90 papers in peer-reviewed journals and several book chapters. She recently published a review in a special issue of the Journal of Invertebrate Pathology on the diversity of large invertebrate DNA viruses.

Dieter Palmberger
Senior Scientist, Austrian Center of Industrial Biotechnology (ACIB)
Baculovirus-Based Manufacturing of Large Biomolecular Assemblies in Trichoplusia ni Cells

Abstract

Large biomolecular assemblies such as viruses and virus-like particles (VLPs) are next generation biopharmaceuticals used in regenerative medicine as delivery vehicles and in preventive medicine as novel vaccines. Furthermore, cancer therapy and neurosurgery are applications for such products. Current technologies for production are more than 50 years old and the quality of the vaccines produced is debated. The baculovirus-insect cell system has proven to be a very powerful tool in the field of recombinant protein expression technology. To date, for the generation of many products, including at large-scale, mostly Sf9 cells are being used. Recently, new production cell lines derived from Trichoplusia ni have been developed, as they are free from contaminating virus, highly feasible for serum-free cell culture, and superior for high yield production of secreted proteins. Nevertheless, little is known about process engineering of Trichoplusia ni cell-based production processes. Further bottlenecks are reduced yield induced by stress response to baculovirus infection, differences in glycosylation patterns, and proper purification schemes that guarantee consistent cell and baculovirus-free products, especially in the case of secreted VLPs. In this project we gained a deeper knowledge of Trichoplusia ni cell physiology and developed several strategies in order to overcome the stated bottlenecks of Trichoplusia ni cell-based production processes.

Biography

Dieter Palmberger studied biotechnology and obtained his master’s degree in 2007. Afterwards he started to work at the University of Natural Resources and Life Sciences in Vienna dealing with the development of different systems for the baculovirus-based production of recombinant proteins. After he finished his PhD in 2011 he worked as a postdoc on a joint project at the University of Veterinary Medicine in Vienna concerning the insect cell-based production of recombinant glycosidases. In 2013 he moved back to the University of Natural Resources and Life Sciences where he led a project dealing with the glycosylation of insect cell-expressed vaccines. Since 2016 he has been employed at the Austrian Center of Industrial Biotechnology leading a project on the development of a virus vaccine vector platform based on measles virus.

Robert L. Harrison
Research Molecular Biologist, Invasive Insect Biocontrol and Behavior Laboratory
USDA Agricultural Research Service
Baculovirus Expression of Insecticidal Proteins

Abstract

Baculoviruses have been evaluated and deployed as safe, environmentally benign biopesticides for the control of insect pests since the 19th century. However, the relatively long time required for host insects to succumb to baculovirus infection has hindered the acceptance and use of baculovirus-based insecticides. This presentation will summarize efforts to address this drawback through the construction of recombinant baculoviruses that express insecticidal proteins during infection. A wide variety of neurotoxic peptides, degradative enzymes, regulators of host physiological processes, and other proteins with demonstrated or potential insecticidal properties have been evaluated for their capacity to reduce the time required for baculovirus-infected insect larvae to die and to reduce the amount of larval feeding prior to death. Factors influencing the expression of insecticidal proteins have been explored, with a significant focus on the kind of promoter used to drive transcription and the signal peptide used to mediate secretion from infected cells. Because a number of different baculoviruses have been the subject of genetic modification to improve insecticidal activity, the presentation will begin with a description of the diversity among large DNA viruses of insects, both in the family Baculoviridae and in families of related insect viruses.

Biography

Bob Harrison received a PhD in Biochemistry in 1996 from Texas A&M University in the laboratory of Max Summers, with a dissertation on the FP25K “few polyhedra” gene of AcMNPV. He then worked on a number of projects relating to the use of baculoviruses as biopesticides in the laboratory of Bryony Bonning at Iowa State University from 1996 to 2003, followed by research in the laboratory of Don Jarvis at University of Wyoming from 2004 to 2005 on a genetic engineering approach to modifying glycosylation of baculovirus-expressed proteins in lepidopteran larvae. Since 2005, Bob has continued work on the genetic and biological characterization of baculoviruses of significance for insect pest control as a Research Molecular Biologist in the USDA-ARS Invasive Insect Biocontrol and Behavior Laboratory in Beltsville, Maryland.

David A. Theilmann, PhD
Research Scientist, Agriculture and Agri-Food Canada
The Ins and Outs of Baculovirus Infection: Dissecting the
Mechanisms of Nucleocapsid Nuclear Import and Egress

Abstract

The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) produces two virion forms during the infection of insects or cultured cells: budded virus (BV) and occlusion-derived virus (ODV). ODV are assembled in the nucleus and released upon liquefaction of the infected insect, and transmit the virus from host to host in the environment. BVs are produced by budding at the cell surface and are required for systemic infection of a host or for cell to cell transmission in cell culture. The BV virion is consequently critical for most biotechnological applications, from protein expression to gene therapy. It is essential therefore to understand how: 1) BV infects the cell and transports nucleocapsids to the nucleus to initiate infection and 2) how newly synthesized nucleocapsids are transported out of the nucleus to the plasma membrane from which they will bud to form BV. BVs bind and enter cells by receptor mediated endocytosis, and nucleocapsids are transported through the cytoplasm by a process that involves host actin polymerization that is initiated by the nucleocapsid protein P78/83. Actin propelled AcMNPV nucleocapsids interact with nucleo pore complexes (NPCs) and we have found that nucleocapsids are able to enter nuclei in digitonin permeabilized cells in the absence of cytosol and under conditions that blocked the Ran-GTPase cycle. However, blocking of the Arp2/3 complex inhibited nucleocapsid nuclear entry. Reconstitution of purified nuclei with G-actin and Arp2/3 restored nucleocapsid entry into the nucleus. These results support the conclusion that nuclear entry is driven by a distinct role of actin-based motility. In contrast to virion entry, the egress of newly assembled progeny nucleocapsids appears to utilize completely different cellular mechanisms and involves multiple viral proteins, and does not involve NPCs. Nucleocapsids appear to bud from the nuclear envelope followed by transit to the plasma membrane by a mechanism that utilizes the microtubule transport system. The critical steps of entry and egress of AcMNPV nucleocapsids therefore take advantage of two quite separate systems: actin polymerization for retrograde transport and entry through NPCs, and egress by budding through the nuclear membrane followed by transport involving kinesin-1 and microtubules for anterograde transport.

Biography

Dr. Theilmann is a Senior Research Scientist with Agriculture and Agri-Food Canada and an Adjunct Professor with the University of British Columbia. He received his PhD in 1988 from Texas A&M University where he worked on the molecular biology of parasitic wasp polydnaviruses in the laboratory of Dr. Max Summers. In his current position his research interests include baculovirus molecular biology with particular interest in regulation of viral gene expression as well as host-pathogen interactions. He has also been involved in several baculovirus genomic studies, the development of foreign protein expression systems, and the development of baculovirus biopesticides.

M. Javad Aman, PhD
President and Chief Scientific Officer, Integrated Biotherapeutics, Inc.
Virus-Like Particle-Based Vaccines for Filoviruses Produced in the
Baculovirus Expression System: Opportunities and Challenges

Abstract

Filoviruses cause severe hemorrhagic fever resulting in significant morbidity and mortality in humans. A number of vaccine platforms are in development for filoviruses that include replication-competent and replication-defective virus-vectored technologies, virus-like particles (VLPs), as well as sub-viral particles. We have generated VLPs for Ebola, Sudan, and Marburg viruses using baculovirus mediated expression in Sf9 cells. A process was developed for purification at 2 L scale of two types of VLPs carrying either two (glycoprotein [GP] and VP40) or three (GP, VP40, and nucleoprotein [NP]) filovirus proteins. The VLPs, formulated in QS-21 adjuvant, were used to vaccinate rodents and nonhuman primates (NHPs). Full efficacy was observed upon two immunizations when three viral genes were incorporated into VLPs, while VLPs lacking NP were only partially protective. While a direct correlation between survival and antibody titer against GP was observed, the requirement for NP, a strong inducer of T-cell responses, suggested that optimal vaccine efficacy requires a combination of humoral and cell-mediated immunity. Sf9-produced VLPs were also used as an immunogen in NHPs and several broadly neutralizing monoclonal antibodies were generated. Challenges and opportunities associated with VLPs produced using the baculovirus expression system will be discussed.

Biography

Dr. M. Javad Aman is a molecular immunologist with over 20 years of experience in the field of vaccine and immunotherapeutics development for infectious diseases. Dr. Aman received his PhD at the University of Mainz in Germany and completed two post-doctoral fellowships at the National Institutes of Health (NIH) and University of Virginia with a focus on regulation of B and T-cell responses. He then moved to the US Army Medical Research Institute of Infectious Diseases (USAMRIID), where he focused on the study of filoviruses as well as several bacterial toxins. This research led to the discovery of virus-like particles as potential vaccines and diagnostic tools for filoviruses. Dr. Aman has also been on the forefront of research on staphylococcal toxins and developed several vaccine candidates for this pathogen. Dr. Aman founded the research-based company Integrated Biotherapeutics Inc. (IBT) in 2007 and grew the company to its current size of 30 employees focused on development of vaccines and antibody therapeutics for emerging infectious disease.

Sergei Zolotukhin, PhD
Professor, Division of Cellular and Molecular Therapy, University of Florida
Direct Head-to-Head Evaluation of Recombinant Adeno-Associated Viral (rAAV)
Vectors Manufactured in Human Versus Insect Cells

Abstract

The major drawback of the baculovirus system for rAAV manufacturing is relatively poor infectivity of many serotypes produced in Sf9 cells. In fact, most of the BAC-derived serotypes, with few exceptions, are characterized by altered capsid composition and lower potency. Hereby, we describe a significantly upgraded OneBac system incorporating a modified cap helper gene encoding adjustable ratios of VP1:VP2:VP3 capsid proteins. The ratio could be fine-tuned by utilizing a serotype-specific attenuated Kozak sequence and leaky ribosome scanning. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side-by-side with HEK 293 derived vectors. The re-designed rAAVs are characterized by significantly higher biological potencies, even in a comparison to HEK 293-manufactured rAAVs mediating, in the case of rAAV5, four-fold higher transduction of brain tissues in mice. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing (NGS), demonstrating significantly lower levels of collaterally packaged contaminating DNA for rAAV5 produced in Sf9 cells. Thus, the latest version of the OneBac system yields rAAV vectors of superior infectivity and exceptional purity, providing a scalable platform for GMP-grade vector production.

Biography

Sergei Zolotukhin graduated from Kiev State University, Ukraine and pursued his graduate studies and PhD at the Institute of Molecular Biology and Genetics, Ukraine. His postdoc was completed at the State University of New York, USA, and he is now Associate Professor in the Department of Pediatrics at the University of Florida (UF), USA. Dr. Zolotukhin’s research program comprises two goals: to develop a better recombinant adeno-associated virus (rAAV) vector for tissue-specific delivery of therapeutic transgenes, and to develop gene based therapies for obesity. He has authored more than 80 publications in refereed journals as well as 12 patents. His track record includes 18 uninterrupted years of multiple NIH funding.

Linda A. King, PhD
Pro Vice-Chancellor, Research and Global Partnerships; Professor of Virology
Oxford Brookes University
BacMam Expression Vectors: Applications in Gene Therapy

Abstract

BacMams are modified baculovirus vectors that contain promoters for gene expression in mammalian cells. BacMams have proven to be efficient for gene delivery into a wide variety of mammalian cells and provide an alternative to other plasmid- and viral-based expression systems. Construction of BacMam vectors is straightforward, using standard protocols to generate recombinant baculoviruses with modified transfer vectors that contain a mammalian gene expression cassette. This presentation will focus on a potential application of BacMam technology in gene therapy associated with organ transplantation together with recent advances to enhance the BacMam system.

Ischemia-reperfusion injury (IRI) is the main cause of delayed graft function in kidney transplantation. Cellular damage from IRI results from reduced adenosine triphosphate (ATP) levels and increases in reactive oxygen species. The application of manganese superoxide dismutase (SOD) has been shown to ameliorate IRI damage. We have developed an in vitro model to simulate IRI in a variety of kidney cell lines and have shown that overexpressing sod-2 using a BacMam vector results in a significant increase in ATP levels and reduction in the release of lactate dehydrogenase from injured cells. We have also shown that BacMam vectors can transduce cells throughout an entire porcine kidney using GFP expression as a reporter gene. Ex vivo delivery of sod-2 significantly increased ATP levels and tissue viability in organs after 24 h of cold perfusion suggesting a potential for BacMam as a gene delivery system for attenuating injury after cold organ preservation.

Biography

Professor Linda King completed her DPhil at the University of Oxford in 1985 and joined Oxford Brookes University in 1986, establishing the Insect Virus Research Group. Linda spent a sabbatical period in the USA with the late Lois Miller in 1994 and was appointed to her current position as Pro Vice Chancellor Research in 2015. She is co-founder of Oxford Expression Technologies Ltd (2007). Current interests include the use of baculovirus vectors for vaccine production and gene therapy as well as continuing a program of basic baculovirus research.

Amine Kamen, PhD
Professor, Department of Bioengineering, McGill University
Critical Assessment of Influenza VLP Production in Sf9 and HEK293 Expression Systems

Abstract

Virus-like particles (VLPs) conformation is a promising platform for antigen design and development of candidate vaccines. In a preliminary study, influenza VLPs were produced in HEK293 suspension cells and Sf9 insect cells. The systems were assessed for their ability to produce influenza VLPs composed of hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1) or Gag protein, and compared from a bioprocessing standpoint by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques. This study and follow-up studies designed to increase the production yields of enveloped VLPs in both systems highlight key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

Biography

Amine Kamen is Professor of Bioengineering at McGill University, and Canada Research Chair in Bioprocessing of Viral Vaccines. He is Researcher Emeritus of the National Research Council of Canada (NRC) where he was employed until early 2014 as head of the Process Development section of the Human Health Therapeutics Portfolio. At NRC, he established one of North America's largest and most advanced governmental centers for animal cell culture addressing process development and scale-up of biologics. Also, along with his team, he developed and licensed multiple technology platforms for efficient manufacturing of recombinant proteins and viral vectors and vaccines to industry and led technology transfer to manufacturing sites for clinical evaluation and commercialization.

His current research activities focus on uncovering mechanisms associated with cell production of viral vectors and viral vaccines; cell and metabolic engineering; process control and monitoring; and process analytical technologies of high-yield productions of viral vectors for gene delivery and vaccination. He has published over 130 papers in refereed international journals and acts as a consultant for several national and international private and public organizations.

Gale E. Smith, PhD
Vice President of Vaccine Development, Novavax, Inc.
Challenges in the Development of a Zika Virus Envelope Dimer (EnvD)
Vaccine in Insect Cells

Abstract

Zika virus (ZIKV) is a flavivirus responsible for an ongoing global epidemic, including reports of sexual and mosquito-to-human transmission in the United States. Zika disease is associated with fetal microcephaly, as well as other birth and developmental defects. There remains an urgent medical need for the development of a safe and effective ZIKV vaccine.

The development of flavivirus subunit protein vaccines based on the major viral envelope (E) glycoprotein, the principle target for neutralizing antibodies, has been technically challenging. Described is the use of the baculovirus – Sf9 insect cell system to express a soluble ZIKV Envelope Dimer (ZIKV EnvD) that is correctly folded and with acceptable yields. ZIKV EnvD was engineered to contain an additional N-linked glycosylation site and co-expressed in Sf9 cells with ZIKV prM and specific chaperones to produce secreted ZIKV E homodimers. ZIKV EnvD binds with high avidity to Zika-specific human convalescent serum and the human receptor DC-SIGN. Biophysical and immunological characterization of EnvD confirmed the presence of a highly conserved, cross-neutralizing, quaternary envelop dimer epitope (EDE1). Dengue EDE1 mAb binds with high affinity to ZIKV EnvD and the bases for a potency assay for the vaccine. ZIKV EnvD vaccine plus a saponin Matrix-M™ adjuvant induces antibodies in mice and non-human primates that neutralize ZIKV strains from both Brazil and Puerto Rico and dengue serotypes 2 and 4. Phase I clinical trials are forthcoming.

Clifton E. McPherson, PhD
Vice President, Regulatory CMC, Protein Sciences Corporation
Production of Recombinant Hemagglutinin for Flublok at the 20,000 L Scale

Biography

Dr. McPherson is Vice President of Regulatory CMC at Protein Sciences Corporation. He joined Protein Sciences in 2005 as Scientist and Project Manager in Process Development, where he was manager of a National Institutes of Health (NIH) funded SARS vaccine project. Dr. McPherson then served as Director and Senior Director of Quality Control, Senior Director of Formulation and Process Analytics, and Vice President of Product Development. Prior to joining Protein Sciences, Dr. McPherson was an Instructor at the University of Connecticut Health Center following postdoctoral fellowships at Brown University and the University of Connecticut Health Center. He holds a PhD in Molecular Biology from Vanderbilt University.

Robin Levis, PhD
Deputy Director, Division of Viral Products, FDA CBER
A Regulatory Perspective on Baculovirus Expression Systems

Biography

Dr. Levis is the Deputy Director of the Division of Viral Products in the Office of Vaccines Research and Review in the Center for Biologics Evaluation and Research, US Food and Drug Administration. Dr. Levis joined the FDA in 1995, after working in NCI, NIH from 1988–1992 and USUHS, Bethesda Naval Hospital from 1992–1995 where she worked on papillomavirus replication and coronavirus pathogenesis, respectively. In 1995, Dr. Levis joined the Laboratory of Vector Borne Viral Disease at FDA and worked on the role of the dengue virus nonstructural protein 1 (NS1) in virus replication. Shortly after joining the lab, Dr. Levis took over the review of all rabies virus vaccines. In addition, she expanded her research efforts to include a collaborative study with industry on the development of an alternative potency test for rabies vaccines. In addition to her research and regulatory contributions to the review of rabies virus vaccines, Dr. Levis’ regulatory responsibilities included the review of Investigational New Drugs and Biological License Applications related to the development and licensure of both papillomavirus vaccines. She served as the chair of the BLA review committee for the licensure of Cervarix, comprised of papillomavirus L1 protein expressed from recombinant baculoviruses in Hi5 insect cells. Dr. Levis served as the regulatory coordinator for the Division of Viral Products from 2002–2008. In this position, she was responsible for managing the regulatory review process for all viral vaccine products. She has been the Division Deputy Director since 2008. Dr. Levis received her PhD in Microbiology from Washington University, St. Louis, Missouri.

Tsafi Danieli, PhD
Head of Protein Expression Facility, The Hebrew University of Jerusalem
Reproducing Irreproducible Results: Lessons Taught from a Self Destructing Protein


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