Baculovirus Titers Research Articles

Production of adeno-associated virus in insect cells using multiple gene deleted baculovirus

Adeno-associated virus (AAV) is one of the most frequently used viral vectors in the field of gene therapy. However, the industrial production of AAV is facing key bottlenecks such as low yield and high-cost. The aim of this study was to establish a technology system for production of AAV in the double virus infected insects by using multiple-gene deleted baculovirus. First, a multiple gene deleted baculovirus for AAV production was constructed, and the baculovirus titer and its effect on infected cells was examined. Subsequently, the insect cells were co-infected with the double baculovirus and the infection conditions were optimized. At the final stage, we performed AAV production based on optimized conditions, and evaluated relevant parameters including production titer and quality. The results showed that the titer of AAV produced in the multiple gene deleted baculovirus was not different from that of the wild type, but the rate of cell death was significantly slower upon infection. Using the double virus route for optimized production of AAV, the genome titers were 1.63×1011 VG/mL for Bac4.0-1 and 1.02×1011 VG/mL for Bac5.0-2, which were elevated 240% and 110%, respectively, compared with that of the wild-type. Electron microscopy observations revealed that all three groups exhibited normal AAV viral morphology and they showed similar transduction activity. Taken together, we developed an AAV production system based on the infection of insect cells using multiple-gene deleted baculovirus, which significantly improved the virus yield and showed application potential.

Development of gp64 Gene based Real-time Quantitative PCR Assay for Rapid and Accurate Determination of Baculovirus Titer

Development of gp64 Gene based Real-time Quantitative PCR Assay for Rapid and Accurate Determination of Baculovirus Titer

Rapid Two-Day Baculovirus Titering Using Flow Cytometry.

This chapter outlines a rapid detection method to determine the virus titer of yourbaculovirus stock. Staining of cells with fluorescently labeled gp64 antibody allows for flow cytometer-based quantitation of baculovirus-infected insect cells. In this assay, Sf9 cells are infected with tenfold serial dilutions of the test virus stock, and baculovirus titers are calculated based on the ratio of infected to uninfected cells 13 to 18h after inoculation.

Evaluation of an inducible knockout system in insect cells based on co-infection and CRISPR/Cas9.

Due to comparably high product titers and low production costs, the baculovirus/insect cell expression system is considered a versatile production platform in the biopharmaceutical industry. Its excellence in producing complex multimeric protein assemblies, including virus-like particles (VLPs), which are considered promising vaccine candidates to counter emerging viral threats, made the system even more attractive. However, the co-formation of budded baculovirus during VLP production poses a severe challenge to downstream processing. In order to reduce the amount of budded baculovirus in the expression supernatant we developed an inducible knockout system based on CRISPR/Cas9 and co-infection with two baculoviral vectors: one bringing along the Cas9 nuclease and the other one having incorporated the sequence for sgRNA expression. With our set-up high titer viruses can be generated separately, as only when both viruses infect cells simultaneously a knockout can occur. When budding essential genes gp64 and vp80 were targeted for knockout, we measured a reduction in baculovirus titer by over 90%. However, as a consequence, we also determined lower overall eYFP fluorescence intensity showing reduced recombinant protein production, indicating that further improvements in engineering as well as purification are required in order to ultimately minimize costs and timeframes for vaccine production utilizing the baculovirus/insect cell expression system.

Impact of Group II Baculovirus IAPs on Virus-Induced Apoptosis in Insect Cells.

Apoptosis plays an important role in virus-host interactions and is a major element of the insect immune response. Exploring the regulatory mechanisms of virus-induced apoptosis through the expression of apoptotic genes holds important research and application value. Functional research on the reported inhibitor of apoptosis proteins (IAPs) mainly focuses on the group I baculovirus, while the functions of the group II baculovirus IAPs remains unclear. To explore its role in the regulation of the apoptosis of insect cells, we constructed the transient expression vector (pIE1 vectors) and the recombinant baculovirus expressing Bsiap genes (from the Buzura suppressaria nucleopolyhedrovirus) of the group II baculovirus. Apoptosis gene expression results and the virus-induced apoptosis rate show that the overexpression of BsIAP1 could promote apoptosis in insect cells. However, the overexpression of BsIAP2 and BsIAP3 decreases the expression of apoptotic genes, revealing an inhibitory effect. Results on the impact of baculovirus-induced apoptosis also confirm that BsIAP1 reduces viral nucleocapsid expression and the baculovirus titer, while BsIAP2 and BsIAP3 increase them significantly. Furthermore, compared with single expression, the co-expression of BsIAP2 and BsIAP3 significantly reduces the rate of virus-induced apoptosis and improves the expression of nucleocapsids and the titer of offspring virus, indicating the synergistic effect on BsIAP2 and BsIAP3. In addition, combined expression of all three BsIAPs significantly reduced levels of intracellular apoptosis-related genes (including apoptosis and anti-apoptosis genes), as well as apoptosis rate and progeny virus titer, indicating that life activities in insect cells are also inhibited. These findings reveal the relationship between apoptosis and group II baculovirus IAP, which provide an experimental and theoretical basis for further exploration of the molecular mechanism between group II baculoviruses and insect cells.

Establishment, verification and application of rapid detection of baculovirus infectious titer by flow cytometry

Titer detection of baculovirus usually is time-consuming. It is important to establish a rapid detection method for baculovirus titer. In this report, Staining of cells with a fluorescently labeled anti-gp64 antibody allows for identification of infected insect cells. By inoculating cultures with a series of log dilutions of virus, and staining of the cultures 13–22 h post inoculation, the ratio of infected to un-infected insect cells can be determined by flow cytometry(FCM). Statistical analysis of the percentage of infected cells in the virus dilution series enables accurate infectious titer determination.The culture time, cell growth state, the concentration of GP64-APC antibody and the concentration of inactivated FBS in diluent were optimized. The generality, repeatability and intermediate precision of the method were verified. The FCM method has the advantages of simplicity, accuracy, low cost and good repeatability.

Purification of baculovirus vectors using heparin affinity chromatography

Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications.

Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.

Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

Suspension culture titration: A simple method for measuring baculovirus titers

The baculovirus-insect cell expression system is an important technology for the production of recombinant proteins and baculovirus-based biopesticides. Budded virus titration is critical when scaling up baculovirus production processes in suspension cultures, to ensure reproducible infections, especially when a low multiplicity of infection (MOI) is applied. In this study, a simple suspension culture titration (SCT) assay was developed that involves accurate measurements of the initial cell densities (ICDs) and peak cell densities (PCDs) of an infected culture, from which the MOI and hence the virus inoculum infectious titer can be estimated, using the established Power-Nielsen baculovirus infection model. The SCT assay was assessed in parallel with two adherent culture-based assays (MTT and AlamarBlue) for the Heliothine baculovirus HaSNPV, and was shown to be more objective, time-efficient and reproducible. The model predicted a linear correlation between log(PCD/ICD) and log(MOI), hence an alternative model-independent SCT assay was also developed, which relies on a well-replicated standard curve relating suspension culture-derived PCD/ICD ratios with plaque or endpoint assay-derived MOIs. Standard curves with excellent linearity were generated for HaSNPV and the industrially significant rAcMNPV, demonstrating the feasibility of this simple titration approach, especially in terms of its applicability to a wide range of virus infection kinetics.

Determination of Recombinant Baculovirus Display Viral Titer

The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. In addition to easy cloning techniques and abundant viral propagation, the system's insect cell environment provides eukaryotic post-translational modification machinery. The baculovirus display vector system provides a number of advantages over prokaryotic systems, allowing the combination of genotype with phenotype, enabling presentation of foreign peptides or even complex proteins on the baculoviral envelope or capsid. Baculoviruses permit larger gene insertions, are easily propagated, and can be grown to high titers. Furthermore, surface modifications of the viral capsid enable specific targeting. This strategy can be used to enhance viral binding and entry to a wide variety of both dividing and nondividing mammalian cells, as well as to produce antibodies against the displayed antigen. In addition, the technology should enable modifications of intracellular behavior, i.e., trafficking of recombinant "nanoparticles," a highly relevant feature for studies of targeted gene or protein delivery. Although baculovirus titer can be determined by standard methods such as classical plaque assays or end-point dilution assays, such methods often are tedious and time-consuming. The protocol described here is rapid and can be performed directly using marker genes such as green fluorescent protein or beta-galactosidase regulated by baculovirus-specific promoters, or indirectly as an immunoassay with baculovirus-specific antibodies (e.g., anti-gp64).

A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line

A new rapid method for titrating baculovirus stocks has been developed using a novel cell line Sf-9 Easy Titer (Sf-9ET). The Sf-9ET cell line has been transfected with plasmid DNA containing the enhanced green fluorescent protein (eGFP) gene under the control of the baculovirus polyhedrin promoter. When used in the titration assay, the Sf-9ET cells turn green when they are infected with baculovirus due to the activation of the polyhedrin promoter/eGFP complex by baculovirus gene products expressed during the infection. Using a 96-well plate format end-point dilution assay, baculovirus titers can be determined in three days using a fluorescence microscope.

Rapid Titer Determination of Baculovirus by Quantitative Real-Time Polymerase Chain Reaction

Titer determination is a prerequisite for the study of viruses. However, the current available methods are tedious and time-consuming. To improve the efficiency of titer determination, we have developed a rapid and simple method for the routine detection of baculovirus titers using a quantitative real-time PCR. This method is based on the amplification of approximately 150-bp fragments located in the coding regions of selected genes. The PCR was found to be quantitative in a range of 10(3) to 10(9) virus particles per 200 microL of supernatant, and the results were closely correlated with titers detected from 50% tissue culture infectious doses (TCID(50)) of baculovirus. This quantitative real-time PCR requires only 30 min to perform, and the entire titer determination can be accomplished within 1 h without the need for cell seeding or further virus dilution and infection. Because this technology is easy to operate, generates data with high precision, and most importantly is very quick, it will certainly be broadly applied for titer determination of baculoviruses in the future.

Utilization of Nucleotide Probes in Elisa Procedure for the Quantitative Determination of Baculovirus Titer

The present work is the development of a simple, rapid, and universally applicable titration method that involves the direct detection of the viral DNA‐binding protein (DBP) gene derived from Autographa californica nucleopolyhedrovirus (AcNPV DBP) as a target for quantitative titer determination of baculovirus and the use of biotin specific probes directed to viral DBP gene. The procedure entails the amplification of the AcNPV DBP gene by using the PCR technique in the presence of digoxigenin‐11‐dUTP from the negative control (non‐infected) and infected samples, and the synthesis of the specific biotin label nucleotide probes directed to the AcNPV DBP gene. These specific probes are then used in the Enzyme Linked Immunosorbent Assay (ELISA) using the immobilized streptavidin on polystyrene microtitration plates for the quantitative determination of baculovirus titer. The plot of O.D.λ=405 nm against the log of the titer (pfu/mL) generated a straight line. The linear range for titer determination of baculovirus was between 102 and 105 pfu/ml−1 for 50 µl of supernatant. Moreover, because of the 96% homology of sequences concerning DBP gene between Bombyx mori nucleopolyhedrovirus (BmNPV) and AcNPV, specific biotin label nucleotide probes obtained from this procedure could be used as a universal tool for titer determination of both viruses.

Estimation of baculovirus titer based on viable cell size

In this paper, a simple and rapid protocol for determination of baculovirus titers based on increasing viable insect cell size/diameter following virus infection is presented. There are different methods available for determining virus titers such as plaque assays end-point dilution, quantitative real-time polymerase chain reaction and flow cytometry. However, most of these methods are time consuming and labor intensive. The titer estimation method presented here can be completed in approximately 28 h from start to finish. In this method, the Vi-CELL (Beckman Coulter) was used to measure cell diameter change over a range of virus dilutions, following infection. The cell diameter change data were used to compute the virus titer using a statistical method called the method of moments that we have described previously.

Selection of Peptide Ligands Specific for Baculovirus DNA‐Binding Protein from the Flitrx™ Random Peptide Display Library

Autographa californica nucleopolyhedrovirus (AcNPV) is a baculovirus that is widely employed as a vector for the expression of foreign genes and pest control. Although baculoviruses, including AcNPV, efficiently replicate in the nuclei of arthropod cells, the dynamics and mechanism of DNA replication within the infected cell are still poorly understood. It has been found that the DNA‐binding protein (DBP) is an early gene product and appears to be crucial for viral DNA replication. Presented here is the selection of peptide ligands that specifically bind to DBP for AcNPV from the FliTrx™ random peptide display library; this entails the amplification, cloning of the DNA‐binding protein (DBP) gene from AcNPV and the construction of the expression plasmid for DBP, and the expression and purification of the recombinant His.Tag AcNPV DBP that was used as a target molecule for the selection of the peptide ligands specific for AcNPV DBP. The affinity and efficiency of such peptide ligands were then measured by ELISA procedures. The beneficial aspect of this research is the monospecificity quality of the peptide ligands specific for AcNPV DBP. They could be used for the study of the dynamics of the viral genome and its replication within the infected cell, for the development of a quantitative method for the determination of the presence of baculovirus in various samples, for the development of a peptide ligandbased assay for the determination of baculovirus titers; or they could be immobilized on a chromatographic support for an improved affinity purification of AcNPV DBP.

A rapid method for estimation of baculovirus titer based on viable cell size

Baculovirus protein expression system is a powerful tool for producing recombinant proteins. To optimize conditions for efficient recombinant protein expression, it is important to determine titer of virus stock for arriving at an optimal multiplicity of infection (MOI) that maximizes recombinant protein expression. Traditionally plaque assays have been used for titer determination. Other methods such as endpoint dilution, quantitative real-time polymerase chain reaction and flow cytometry have been developed to aid the determination of virus titers. However, most of these methods are time-consuming and labor intensive. In this regard, a simple and rapid method for determination of virus titers based on the cytopathic effects that lead to viable cell size increase following virus infection is presented in this paper. In this study, the Vi-CELL (Beckman Coulter) was used to measure cell-diameter over a range of virus dilutions, following infection. Applying statistical modeling techniques, the viable cell-diameter data was used to estimate the virus titer. The results indicated that the viable cell-diameter based titer estimation to be reliable and comparable to titers determined by the traditional plaque assay.

A Flow Cytometric Assay for Rapid, Accurate Determination of Baculovirus Titers

A Flow Cytometric Assay for Rapid, Accurate Determination of Baculovirus Titers

Development of an antibody-based assay for determination of baculovirus titers in 10 hours.

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.

Concentration of recombinant baculovirus by cation-exchange chromatography.

BioTechniquesVol. 26, No. 5 BenchmarksOpen AccessConcentration of Recombinant Baculovirus by Cation-Exchange ChromatographyJames BarsoumJames Barsoum*Address correspondence to Dr. James Barsoum, Biogen, Inc., 12 Cambridge Center, Cambridge, MA 02142, USA. Internet: E-mail Address: jim_barsoum@biogen.comBiogen, Cambridge, MA, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99265bm07AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByProduction of Baculovirus and Stem Cells for Baculovirus-Mediated Gene Transfer into Human Mesenchymal Stem Cells22 September 2020Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus SystemExpression and purification of recombinant feline interferon in the baculovirus-insect larvae systemProcess Biochemistry, Vol. 49, No. 6Virus Separation Using Membranes20 November 2013Membrane Supported Virus Separation from Biological Solutions14 June 2013 | Chemie Ingenieur Technik, Vol. 85, No. 8Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatographyJournal of Virological Methods, Vol. 183, No. 2Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: Current advancesBiotechnology Advances, Vol. 29, No. 6Nonintegrating DNA Virus25 October 2011Baculovirus as vectors for human cells and applications in organ transplantationJournal of Invertebrate Pathology, Vol. 107Quality control and analytical methods for baculovirus-based productsJournal of Invertebrate Pathology, Vol. 107Requirements for baculoviruses for clinical gene therapy applicationsJournal of Invertebrate Pathology, Vol. 107Development and validation of a HPLC method for the quantification of baculovirus particlesJournal of Chromatography B, Vol. 879, No. 1High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization12 June 2009 | BMC Biotechnology, Vol. 9, No. 1Enhancing the multiplication of nucleopolyhedrovirus in vitro by manipulation of the pHJournal of Virological Methods, Vol. 161, No. 2Purification of recombinant baculoviruses for gene therapy using membrane processes2 April 2009 | Gene Therapy, Vol. 16, No. 6Concanavalin a affinity chromatography for efficient baculovirus purificationBiotechnology Progress, Vol. 23Virus Production for Clinical Gene Therapy24 February 2009Targeting and Purification of Metabolically Biotinylated BaculovirusHuman Gene Therapy, Vol. 19, No. 6Baculovirus-Infected Insect Cells Expressing Peptide-MHC Complexes Elicit Protective Antitumor Immunity20 December 2007 | The Journal of Immunology, Vol. 180, No. 1Baculovirus envelope fusion proteins F and GP64 exploit distinct receptors to gain entry into cultured insect cellsJournal of General Virology, Vol. 88, No. 12Ion-Exchange Membrane Chromatography Method for Rapid and Efficient Purification of Recombinant Baculovirus and Baculovirus gp64 ProteinHuman Gene Therapy, Vol. 18, No. 7High yield purification of functional baculovirus vectors by size exclusion chromatographyJournal of Virological Methods, Vol. 142, No. 1-2Sampling Viruses from Soil14 May 2007Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus SystemBaculovirus Vectors for Gene TherapyBaculovirus as a highly efficient expression vector in insect and mammalian cellsActa Pharmacologica Sinica, Vol. 26, No. 4Highly efficient baculovirus-mediated gene transfer into rat chondrocytes1 January 2004 | Biotechnology and Bioengineering, Vol. 88, No. 5Generation of chimeric baculovirus with histidine-tags displayed on the envelope and its purification using immobilized metal affinity chromatographyEnzyme and Microbial Technology, Vol. 33, No. 4Baculovirus as Mammalian Cell Expression Vector for Gene Therapy: An Emerging StrategyMolecular Therapy, Vol. 6, No. 1A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus22 June 2001 | Gene Therapy, Vol. 8, No. 11Estimation of Baculovirus Titer by β-Galactosidase Activity Assay of Virus PreparationsTetsuro Yahata, Stephanie Andriole, Kurt J. Isselbacher & Toshi Shioda29 August 2018 | BioTechniques, Vol. 29, No. 2Application of Recombinant Baculoviruses in Biopharmaceutical Research Vol. 26, No. 5 Follow us on social media for the latest updates Metrics Downloaded 370 times History Published online 22 August 2018 Published in print May 1999 Information© 2018 Author(s)PDF download

Optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells.

We have previously described a quantitative model of the trafficking of baculovirus in insect cells that considers the various infection steps such as attachment, internalization, endosomal fusion, and nuclear accumulation. Concepts from the model were used to design synchronous infection regimens for various cell lines, to analyze the inherent inefficiency of existing assays for virus titer, and to develop a modified end-point dilution assay optimized to measure more completely the concentration of intrinsically infectious particles in a virus inoculum. The titer obtained from existing assays incompletely counts infectious virus particles due primarily to the incomplete adsorption of the virus during the short, standard 1-h incubation period. For representative assays, the calculated bound virus is generally about 10% of the added virus, but could be as low as 1.4%, underestimating actual titers by 3-70-fold. A modified end-point dilution assay involving centrifugation has been developed from both quantitative and qualitative analyses. The ratio of particle to plaque-forming unit with the optimized assay was 4-6 compared to 100-300 for typical assays, representing a significant improvement in the measurement of total infectious virus concentration.