Address correspondence to B.A. Merrick, NIEHS, PO Box 12233, Research Triangle Park, NC 27709 USA.
This report summarizes the progress and applications of the baculovirus expression vector system (BEVS) in insects. An International Baculovirus and Insect Cell Gene Expression Conference was held 26-30 March 1995 in Pinehurst, North Carolina, to share and disseminate research in this emerging field of biotechnology. More than 200 people from 22 countries participated in the conference, which was sponsored by American Cyanamid, Glaxo Inc. Research Institute, and 9 other biotechnology companies. The conference consisted of four sessions: baculovirus biology, expression technology, host systems, and applications. Each session began with a historical review of the topic, followed by a series of lectures by invited speakers and short presentations by selected conference participants, with poster presentations in topical areas. Research presented focused on the applications in environmental, human, and animal health, and the prospective uses of baculovirus technology to produce safe biopesticides as replacements for chemical insecticides for crop protection or to design and synthesize recombinant biopharmaceutical products for the treatment of human disease.
The baculovirus expression vector is a helper-independent, eukaryotic DNA viral vector that infects lepidopteran (butterflies and moths) insects and insect cells. Approximately 500 recombinant genes have been expressed in the BEVS under the transcriptional regulation of single or multiple baculovirus promoters representing genes of immediate early, delayed early, late, and very late promoter classes, the most common being the polyhedrin promoter. An advantage of BEVS over bacterial expression systems is that BEVS expressed proteins are often post-translationally modified similar to mammalian cells, which is an important factor for biological activity and protein function studies. Ninety-five percent of expressed recombinant proteins are biologically active. The prototype baculovirus is the Autographa californica nuclear polyhedrosis virus (AcNPV). The three most popular insect cell lines used in the BEVS are Sf9 and Sf21 from the fall armyworm, Spodoptera frugiperda, and TN5B1-4 (High 5) from the cabbage looper, Trichoplusia ni. High 5 cells typically exhibit greater recombinant protein synthesis than Sf9 or Sf21. However, the high metabolic activity of this cell line results in a higher proportion of by-product accumulaton.
In the natural environment, when infected lepidopteran larvae (caterpillars) die, polyhedrin encapsulated baculovirus particles left in the decomposing tissue contaminate adjacent plants. Uninfected larvae feed on the contaminated plants, ingest the polyhedra, and become infected. Infection begins in the midgut, where the polyhedrin protein dissolves in the alkaline environment and progresses through the larvae via the tracheal system. Recent studies have revealed that baculovirus DNA replication triggers apoptosis, the host's primary defense, which results in an abortive infection at both the cellular and organismal level. The ability to block cellular apoptosis would greatly increase the infectivity and host range of the baculovirus and increase recombinant protein production and stability. The AcNPV encodes two genes to accomplish this: p35 and iap. The human proto-oncogene bcl-2 also inhibited insect cell apoptosis when co-expressed with recombinant protein.
Plant and Agricultural Research
The BEVS has tremendous potential in agriculture as an environmentally safe biopesticide against lepidopterans, which cause massive amounts of damage to crops and ornmental vegetation. Several recombinant proteins were reported at this meeting to have been successfully expressed in whole insects using baculovirus vectors, to study insect metabolism, improve post-translational modification and localization, and to synthesize large quantities of the recombinant protein. The success of these studies indicates that the baculovirus could be safely dispersed on crop surfaces and produce a fatal infection after ingestion by lepidopteran larvae in agricultural crop fields. There were several presentations at this conference by scientists engaged in agrichemical BEVS research describing progress toward a marketable biopesticide. The parameters to consider in evaluating new pesticides include efficacy, host range of the product, environmental stability, production, formulation, cost, and marketing. The AcNPV infects only lepidopterans; it is completely harmless to all vertebrates and other invertebrates. The wild-type virus alone has a deleterious effect on the host insects and can reduce crop destruction to some degree. Feeding by the infected host is inhibited only during the terminal stages of infection. Removing the viral genes that act to promote feeding activity and prolong the life of the insect would improve the efficacy of the wild-type virus by killing the host faster. The AcNPV
EGT gene is an example of this class of genes. Field trials are in progress using an EGT-deleted (EGTDEL) construct. Insertion of a species-specific, lethal gene into the virus genome increases both the efficacy and the specificity of the system. The recombinant virus is sprayed on the crop and subsequently ingested by the larvae. As the virus replicates and the infection progresses in the host, the deleterious, recombinant protein is synthesized and metabolized by the insect. The baculovirus becomes a delivery system for the toxic gene, which kills the host more quickly than the wild-type virus. The most commonly studied recombinant toxic gene product is AaIT, a 70-amino acid insect-selective, scorpion protein that induces contractile paralysis in the prey by binding to the voltage site on sodium channels. When cloned in AcNPV, AaIT showed a 38% improvement in speed of action compared to the wild-type strain and gave significantly enhanced control of larval feeding damage in whole plant studies. In
in vitro experiments, an EGT-deleted-AaIT inserted construct (vEGTDEL-AaIT) was the most effective biopesticide against lepidopteran cells. Field tests using vEGTDEL-AaIT AcNPV have been proposed and are pending EPA review. Although the wild-type AcNPV is registered with the EPA, baculovirus biopesticides face many barriers before they could be successfully marketed. The wild-type virus is less effective than pyrethrin and more expensive, diminishing the economic incentive for use. Each recombinant construct must undergo government-approved field testing before receiving EPA registration. To be competititve in the future pesticide market, the baculovirus biopesticides must be as economical as chemical insecticides, and public safety concerns about genetically engineered products must be addressed.
Biomolecular Research
The BEVS is a powerful tool for production of large quantities of recombinant mammalian protein for biomolecular research. The biological activity and similarity to native proteins offer great advantage over conventional bacterial expression systems. Secretion signal peptides, nuclear localization signals, chaperone proteins, and catalytic folding proteins, missing in the BEVS, can be cloned into the viral genome and co-expressed with the desired recombinant protein, permitting proper folding, transport, and localization. The post-translational modification which received the greatest attention at this conference was glycosylation. In insect cells, glycosylation, especially processes that result in complex N-linked side chains containing outer chain galactose and/or terminal sialic acid, often produces oligosaccharide sequences that are not identical to those in the respective native proteins. The theory presented was that following induction of the glycosyltransferase enzymes during infection, glycosylation processes switch from the high mannose-type glycan assembly normal for lepidopteran cells to a complex type of oligosaccharide processing. The transition results in a hybrid process and infidelity. Many human proteins have been expressed using the BEVS for basic research, commercial applications, and development of new pharmaceuticals.
Development of stably transformed insect cell lines, in which heterologous genes are integrated into the insect cell genome, offers several advantages over conventional BEVS infection and represents the newest extension of insect cell gene expression technology. The cell lines described at this conference do not experience cell death and lysis associated with viral replication, and they utilize the normal host cell secretory pathways, demonstrate improved secretion and organelle targeting of the recombinant protein, and facilitate the production of expression libraries. Stable expression of foreign genes in Sf9, Drosophila S2, and Aedes albopictus cells has been accomplished, but the recombinant proteins are currently expressed at a lower level than in the BEVS.
Medical Research
The ultimate test of medical research is whether the compound studied or the product developed can be implemented in a clinical setting to benefit the human population. As part of the transition from research laboratory to clinical trial, the drug or chemical must receive approval from the Food and Drug Administration. There are three categories classifying drugs for FDA review (diagnostic, vaccine, and therapeutic) and each has a unique set of requirements, regulations, and restrictions. One of the key elements involved in the expression of diagnostic factors from an expression system is that the recombinant protein must have biological activity comparable to the native protein, yet it must not bind with proteins from the host cells which would contribute to contamination of the recombinant protein preparation. Many insect cell factors, such as localization and secretion signal peptides, or chaperone and folding proteins, do not recognize the recombinant mammalian proteins. Consequently, recombinant secreted membrane and nuclear proteins remain in the cytoplasm of the host cell. Thus, what was a serious liability in the study of nuclear and secreted proteins becomes one of the strongest advantages in the production of diagnostic proteins in BEVS. Auto-antigens, cytochromes, G-protein coupled receptors, and Herpes simplex virus capsid proteins have successfully been produced for diagnostic purposes in BEVS.
The baculovirus expression vector system (BEVS) and its applications. The gene of interest (red capsule) is cloned into the circular DNA of the baculovirus (top). The baculovirus infects Lepidopteran insect larvae or cultured insect cells. Viral replication occurs within the insect host cells, followed by expression of the recombinant gene product (red protein ribbon structure). Potential uses of BEVS proteins are shown for agriculture (lower left) as a biopesticide and in medicine (lower right) as a vaccine or therapeutic.
The development of human and animal vaccines is a second important BEVS medical application. This emerging field was a major focus of discussion at the conference. The first baculovirus-produced human vaccine, based on the immunodeficiency virus (HIV-1) envelope glycoprotein gp160, began human clinical trials in 1987. Since that time, immunogens produced in insect cells from baculovirus vectors have been tested as prophylactic vaccines against acquired immunodeficiency syndrome (AIDS), malaria, respiratory syncytial virus, and influenza, and as therapeutic vaccines for the treatment of AIDS and cancer. Because these are new vaccines produced in BEVS, not recombinant versions of existing vaccines, a direct comparison to earlier forms of the same vaccine is not available. It is too early to determine whether the subjects vaccinated with VaxSyn, the gp160 AIDS vaccine, will remain free of the disease. However, current data from the VaxSyn clincial trials reveal that after vaccination, 100% of the uninfected subjects and 80% of the HIV-infected subjects who had a CD4 > 400 developed an epitope antibody response. Approximately 50% of all subjects (HIV negative and positive) developed neutralizing antibodies. None of the subjects displayed an allergic response to the vaccine, although some did develop a mild, local reaction. For veterinary medicine, a baculovirus-produced vaccine for bluetongue virus (BTV) successfully elicited protective immunity in sheep when the vaccinated sheep were challenged with the homologous virulent bluetongue virus. The global use of vaccines is considered by some medical authorities to be the most cost-effective strategy to prevent disease and death. If the recombinant vaccines now in clinical trials are successful, BEVS and recombinant DNA technology offer the future possibility of new and safer vaccines for a number of diseases.
The class of drugs that faces the greatest difficulty in receiving FDA approval is human therapeutics. To date, no BEVS-generated therapeutic drug has received FDA approval to enter clinical trials, but approval for one drug is imminent. A recombinant membrane CD4 protein, developed at the Center for Blood Research Laboratories at Harvard Medical School, is in final FDA review. The CD4 protein functions as the high-affinity receptor for the HIV envelope glycoprotein, gp 120. The recombinant protein displayed the same biological activity as the native human CD4. The recombinant CD4 was electroinserted in the membrane of red blood cells (RBC-rmCD4) and then placed into the lipid bilayer of liposomes. In vitro, RBC-rmCD4 binds HIV particles, fuses with the HIV envelope, inhibits HIV infection of susceptible target cells and aggregates HIV-infected cells. Another human therapeutic in development reported at the conference is a recombinant glucocerebrosidase (GC), which is used in the treatment of Gaucher's disease, a genetic lipid storage disease. The proposal is to apply polyethylene glycol surface modification (PEGlation) to the GC enzyme to enhance its use in replacement therapy, improve circulation half-life, and reduce immunogenicity.
Before these new drugs can be tested in patients, they must be approved by the FDA. Receiving FDA approval is an intensive process in the development of human therapeutics. The long and complicated review is designed to screen all potential new drugs and to safeguard the public from remedies that have not been adequately researched and tested. As a result, many products do not reach clinical trial. The complexity of the process and the time and costs involved discourage some companies from investing in expensive new drug investigation research. As with all biopharmaceuticals, the production laboratories for BEVS products must meet strict FDA standards for currrent Good Manufacturing Practices (cGMP), careful Standard Operating Procedures (SOP) must be developed and followed for every procedure, and quality control procedures for in-progress and final product testing must be established for quality assurance documentation. BEVS-synthesized pharmaceuticals must meet additional requirements to ensure that an insect-synthesized protein is biologically active and effective against human disease and that there are no insect contaminants that could compromise patient safety. The master virus stock for infecting insect host cells must be qualified; the DNA sequence must be confirmed; and the high titer stock (HTS) must be free of mycoplasma, viruses, and any other contaminants. The titer must be confirmed by plaque assay. The insect host cells must be screened and rigorously maintained in a Working Cell Bank and a Master Cell Bank. The recombinant protein must be highly purified, and all traces of the insect host cells and baculovirus removed. All tissue culture procedures must be highly reproducible. Recombinant technology has often received negative reviews from the media and generated safety concerns from the public, which must be addressed once FDA approval is obtained. The acceptance and success of CD4 and GC will pave the way for a bounty of recombinant biopharmaceuticals in this new technology frontier.
Scale-Up Technology
Commercial BEVS applications in medicine and agriculture will require large quantities of the recombinant protein and thus continued advances in bioengineering for large-scale production; several facets of this technology were presented at the conference. Increasing the volume and mass of a suspension culture is not a linear process, and it presents many logistical problems. Scale-up from conventional 100-mL suspension culture to 36-L stirred reaction vessels and 100-L bioreactors has been successfully accomplished, with no change in the kinetics of cell growth and protein production. The key to successful scale-up in any mechanical system is the harmonious optimization of all components of the system. Because insect cells experience a 30% increase in oxygen uptake immediately after infection, it is usually necessary to agitate the system or to perfuse or inject oxygen into the reaction vessel to prevent anoxia and changes in pH level of the medium. Replacement of growth-limiting nutrients via hollow-fiber microfiltration or a programmed feeding strategy is necesary for efficient metabolism and to prevent excessive by-product accumulation. The multiplicity of infection (MOI) of the high titer stock and the MOI ratios in multiple infections are important kinetic factors, which generated a great deal of discussion among conference participants. Infecting at a low MOI (0.1-0.2) is economical, but allows one population doubling before the cells engulf the virus. This can improve cell health, but it can also cause an asynchronous culture. New advances in bioreactors include a NASA-developed, reduced shear, low turbulence microgravity (0.2 dyne/cm
2) vessel, the high-aspect ratio vessel (HARV), and a dielectrophoresis system (DEP), which uses a nonuniform electric field to remove nonproductive, dead cells from the reactor.
Summary
The BEVS continues to evolve as a powerful, flexible tool for molecular biology, protein function, and biomedical research. Future developments offer the promise of replacement of hazardous chemical insecticides with environmentally safe biopesticides, construction of baculovirus vectors which encode genes for specific post-translational modifications, and establishment of efficient, stably transformed insect cell lines. FDA approval of BEVS-produced products offer the prospect of new biopharmaceuticals, in particular human therapeutics and vaccines, to improve human health and increase the quality of life for millions of people.
