Leslie C. L. Chan

Tunable pores for measuring concentrations of synthetic and biological nanoparticle dispersions

Scanning ion occlusion sensing (SIOS), a technique that uses a tunable pore to detect the passage of individual nano-scale objects, is applied here for the rapid, accurate and direct measurement of synthetic and biological nanoparticle concentrations. SIOS is able to characterize smaller particles than other direct count techniques such as flow cytometry or Coulter counters, and the direct count avoids approximations such as those necessary for turbidity measurements. Measurements in a model system of 210-710 nm diameter polystyrene particles demonstrate that the event frequency scales linearly with applied pressure and concentration, and that measured concentrations are independent of particle type and size. Both an external-calibration and a calibration-free measurement method are demonstrated. SIOS is then applied to measure concentrations of Baculovirus occlusion bodies, with a diameter of ~1 μm, and the marine photosynthetic cyanobacterium Prochlorococcus, with a diameter of ~600 nm. The determined concentrations agree well with results from counting with microscopy (a 17% difference between the mean concentrations) and flow cytometry (6% difference between the mean concentrations), respectively.

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system.

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.

Transcriptome sequencing of and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions

The Heliothine insect complex contains some of the most destructive pests of agricultural crops worldwide, including the closely related Helicoverpa zea and H. armigera. Biological control using baculoviruses is practiced at a moderate level worldwide. In order to enable more wide spread use, a better understanding of cell-virus interactions is required. While many baculoviruses have been sequenced, none of the Heliothine insect genomes have been available. In this study, we sequenced, assembled and functionally annotated 29,586 transcripts from cultured H. zea cells using Illumina 100 bps and paired-end transcriptome sequencing (RNA-seq). The transcript sequences had high assembly coverage (64.5 times). 23,401 sequences had putative protein functions, and over 13,000 sequences had high similarities to available sequences in other insect species. The sequence database was estimated to cover at least 85% of all H. zea genes. The sequences were used to construct a microarray, which was evaluated on the infection of H. zea cells with H. Armigera single-capsid nucleopolyhedrovirus (HearNPV). The analysis revealed that up-regulation of apoptosis genes is the main cellular response in the early infection phase (18 hours post infection), while genes linked to four major immunological signalling pathways (Toll, IMD, Jak-STAT and JNK) were down-regulated. Only small changes (generally downwards) were observed for central carbon metabolism. The transcriptome and microarray platform developed in this study represent a greatly expanded resource base for H. zea insect- HearNPV interaction studies, in which key cellular pathways such as those for metabolism, immune response, transcription and replication have been identified. This resource will be used to develop better cell culture-based virus production processes, and more generally to investigate the molecular basis of host range and susceptibility, virus infectivity and virulence, and the ecology and evolution of baculoviruses.

In vitro production of Helicoverpa baculovirus biopesticides: Automated selection of insect cell clones for manufacturing and systems biology studies

Baculovirus pesticides are increasingly being used as effective biological control agents against caterpillar pests worldwide. Increasing occlusion body (OB) yields per cell in culture is the main challenge to enable commercialization of in vitro production of baculovirus pesticides. Isolating clones from a heterogeneous cell population may allow development of a high virus producing cell clone. To date, the selection of insect clones has been based mainly on laborious cell serial dilution methods which create few viable clones. This work used an automated robotic clone picking system to establish over 250 insect clones of a Helicoverpa zea cell population to be screened for virus production. However, the higher producing clones only produced 10-30% higher OB yields than the original cell population. This study suggested that unless screening of thousands of clones is performed, obtaining a 2-fold increase in OB/cell yield compared to the parent population is unlikely. Nevertheless, it creates pure clones for manufacturing. In addition, two clones that were at least 2-3 times different in OB yields were isolated. Hence, this method can create a high contrast system (OB/cell yield basis), for comparative studies using a systems biology approach, which should inform a more targeted approach to engineer genetically a production cell line.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng. 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids, glucose and yeastolate ultrafiltrate) into Sf 9 cell cultures grown in SF 900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 microg/ml of NS1 at a peak cell density of 4.2 x 10 (6) cells/ml. In contrast, optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 microg/ml of NS1 produced at a peak cell density of 11.3 x 10 (6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10 (6) cells/ml, unless the cultures were stimulated by the addition of 4 microg/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 microg/ml, which was nearly 5-fold higher than optimised batch cultures.

Kinetic characterization of the group II helicoverpa armigera nucleopolyhedrovirus propagated in suspension cell cultures: Implications for development of a biopesticides production process

Large‐scale commercialization of baculovirus biopesticides for the control of insect pests requires a cell culture production process, and knowledge of the infection kinetics is a vital prerequisite for process optimization. Well‐characterized kinetic parameters have so far only been reported for the commercially established recombinant Autographa californica nucleopolyhedrovirus (AcMNPV), a Group I NPV. In this work, key infection kinetic parameters of the Group II NPV Helicoverpa armigera nucleopolyhedrovirus (HaSNPV), and its Few Polyhedra (FP) mutant, were well characterized for the first time, in suspension HzAM1 insect cell cultures, to facilitate the scale‐up of an HaSNPV‐based biopesticide. The FP mutant had a selective advantage over wild‐type HaSNPV in cell cultures, and the kinetic analysis showed that this was due to a superior budding rate, rather than a faster binding rate (BR) or longer budding duration. Another finding was that wild‐type HaSNPV had very poor infection kinetics when compared with AcMNPV, exhibiting an 18‐fold lower BR, a more than 50‐fold lower budding rate, and a 60‐fold lower extracellular/total progeny virus ratio. Such poor infection kinetics have serious implications during scale‐up of an HaSNPV biopesticide production process, including the requirement for large volumes of virus inocula and the difficulty of achieving synchronous infections. Groups I and II NPVs may have very different infection kinetics because of their different envelope fusion proteins. This study is the first to compare the two groups of NPVs in terms of well‐characterized cell‐specific infection kinetics, and the findings may indicate a phylogenetic basis for kinetic differences.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells.

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellularviral components, and the heterologous product titers. The model describes the whole processes of viral infection and theeffect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave goodagreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infectionin batch and fed-batch culture were studied using the model.

PROPERTIES OF A UNIQUE MUTANT OF HELICOVERPA ARMIGERA SINGLE-NUCLEOCAPSID NUCLEOPOLYHEDROVIRUS THAT EXHIBITS A PARTIAL MANY POLYHEDRA AND FEW POLYHEDRA PHENOTYPE ON EXTENDED SERIAL PASSAGING IN SUSPENSION CELL CULTURES

SummarySerial passaging of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) in H. zea (Hz-AM1) insect cell cultures results in rapid selection for the few polyhedra (FP) phenotype. A unique HaSNPV mutant (ppC19) was isolated through plaque purification that exhibited a partial many polyhedra (MP) and FP phenotype. On serial passaging in suspension cell cultures, ppC19 produced fivefold more polyhedra than a typical FP mutant (FP8AS) but threefold less polyhedra than the wild-type virus. Most importantly, the polyhedra of ppC19 exhibited MP-like virion occlusion. Furthermore, ppC19 produced the same amount of budded virus (BV) as the FP mutant, which was fivefold higher than that of the wild-type virus. This selective advantage was likely to explain its relative stability in polyhedra production for six passages when compared with the wild-type virus. However, subsequent passaging of ppC19 resulted in a steep decline in both BV and polyhedra yields, which was also experienced by FP8AS and the wild-type virus at high passage numbers. Genomic deoxyribonucleic acid profiling of the latter suggested that defective interfering particles (DIPs) were implicated in this phenomenon and represented another undesirable mutation during serial passaging of HaSNPV. Hence, a strategy to isolate HaSNPV clones that exhibited MP-like polyhedra production but FP-like BV production, coupled with low multiplicities of infection during scale-up to avoid accumulation of DIPs, could prove commercially invaluable.

Production of Entomopathogenic Viruses

Of all the entomopathogenic viruses, baculoviruses show the most potential to control lepidopteran pests on crops. Many baculoviruses produced in vivo have found commercial success on a range of crops. However, wider use of these highly specific, safe, and environmentally sustainable biopesticides is likely if they can be produced in vitro using readily scalable cell culture processes. In this chapter, the status of knowledge related to baculovirus infections of cells in culture is reviewed in relation to the cell lines used, the media required to grow the cells, and the genetic stability and yield of the viruses following in vitro infections. Helicoverpa armigera nucleopolyhedrovirus is used as a case study to indicate that at current yields an in vitro production process is feasible at the 10,000. l bioreactor scale however, further work is required to resolve quality control issues in production. The application of systems biology to improve yields further and make commercialization more attractive is also addressed.

Genome scale analysis of differential mRNA expression of Helicoverpa zea insect cells infected with a H. armigera baculovirus

Knowledge of baculovirus-insect host interactions at a genome-scale level is important for developing a number of baculovirus-based applications, but the gathering of such knowledge is hindered by the lack of genomic sequences in most insect hosts. In this study, expression kinetics of 24,206 Helicoverpa zea insect transcripts and 134 Helicoverpa armigera nucleopolyhedrovirus (HearNPV) genes at 0, 12, 24 and 48 h post-infection (hpi) were simultaneously analyzed using microarrays, which were developed from sequences obtained by deep transcriptome sequencing. Host genes in pathways important for infection such as those for energy generation, anti-viral peptides, apoptosis, detoxification, DNA polymerase activities, RNA polymerase activities, translation initiation, protein processing and cell cycle arrest were identified. Differential expression was linked to changes in the number of intracellular and extracellular viral genomes and occlusion bodies. The first comprehensive elucidation of HearNPV-H. zea expression kinetics was obtained.