Baculovirus-infected insect cells

A number of allergens from both honey bee and vespid venoms have been cloned and expressed by either Escherichia coli or baculovirus-infected insect cells (table 1) phospholipase Aj [20], hyaluronidase [21], acid phosphatase [13] and Api m6 [14] from honey bee venom, as well as antigen 5 [22], phospholipase A and hyaluronidase [23] from vespid venom, and dipeptidylpeptidases from both bee and Vespula venoms [15, 16]. Their reactivity with human-specific IgE antibodies to the respective allergens has been documented [11-16, 22, 23] and their specificity is superior... 

Production of Core and Virus-Like Particles with Baculovirus Infected Insect Cells... 

In this paper the fundamental aspects of process development for the production of core and virus-like particles with baculovirus infected insect cells are reviewed. The issues addressed include particle formation and monomer composition, chemical and physical conditions for optimal cell growth, baculovirus replication and product expression, multiplicity of infection strategy, and scale-up of the process. Study of the differences in the metabolic requirements of infected and non-infected cells is necessary for high cell density processes. In the bioreactor, the specific oxygen uptake rate (OURsp) plays a central role in process scale-up, leading to the specification of the bioreactor operational parameters. Shear stress can also be an important variable for bioreactor operation due to its influence on cell growth and product expression. 

Viral structural proteins expressed by baculovirus infected insect cells assemble into multimeric structures that resemble viral core-like particles and virus-like particles (CLPs and VLPs, respectively). This presentation has brought the attention of researchers for the potential use of these structures as safe immunological reagents for virus or antibody detection in enzyme immuno assays, as vaccines, and more recently, as gene delivering systems for gene therapy [9]. 

In this paper the fundamental aspects concerning the production of CLPs and VLPs with baculovirus infected insect cells are reviewed. It is not the goal of this communication to review all the aspects of insect cell culture technology, since this would be a task impossible to achieve in a single chapter. The interested reader should refer to the references. This review is structured in four parts ... 

In this part the application of mathematical models to CLP and VLP production with baculovirus infected insect cell cultures is discussed. Special emphasis on model evaluation is made along with the definition of directions in future process development research with this system. 

For the production of CLPs and VLPs with baculovirus infected insect cells the specific proteins that are required for particle formation should be chosen at an early step. The specific particle composition, along with the expression of other non-structural (NS) proteins often has a great impact on particle stability [11] or on particle localisation [12], i.e. - cell-associated or secreted to the supernatant. 

The selection of the monomers to incorporate in the capsid should consider other items along with antigenicity, such as product release by the cells [ 15,24]. Product secretion can have a large impact on overall particle production since baculovirus-infected insect cells often exhibit proteolytic activity, which is mainly intracellular at early times post-infection [25]. The appearance of proteases often coincides with plO and polyhedrin-driven late protein production [26]. 

Table 1. CLPs and VLPs produced with baculovirus-infected insect cells for candidate vaccines...

Optimal conditions for insect cell growth have been extensively studied, but for product expression with a baculovirus infected insect cell the focus should be on the difference in the metabolic requirements of infected vs. uninfected cells, which has been observed to differ after infection. The alanine specific production rate decreases almost four-fold, while phenylalanine specific consumption rate increase 11-fold and glutamine specific consumption decrease [65]. Both an increase [66] and a decrease [67] in glucose consumption rates of insect cells after infection have been reported. This reflects some differences in the media and vectors that were used however, it is normal to expect a higher metabolic burden after infection due to the increase in protein expression rates caused by the infection. This creates a concern about the impact of nutrient limitations on the productivity of the system. 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

The Hu and Bentley model is the only one that tries to describe VLP production and assembling in baculovirus infected insect cells [105]. Nevertheless, regarding VLP assembly, the formalism presented is completely theoretical and based on the assembly pathway of icosahedral viruses. From a process development point of view, this model does not generate enough output to make it applicable to bioreaction operational parameters definition. However, it can be used as the basis for a more structured approach to the VLP assembling process in baculovirus infected insect cells. 

Mathematically modelling of VLP production by baculovirus infected insect cells is not a closed chapter of research, and probably never will be, at least until complete structured models have been developed, taking into account intracellular pools. 

The production of VLPs with baculovirus infected insect cells is entering the phase of industrial application. Due to the complexity of the system a multitude of vector strategies have been developed, ranging from single to fifth-level baculovirus vectors, along with combinations between them. 

Cha, K. Bruel, C. Inglese, J. Khorana, H.C. Rhodopsin kinase Expression in baculovirus-infected insect cells, and characterization of post-transla-tional modifications. Proc. Natl. Acad. Sci. USA, 94, 10577-10582 (1997)... 

Kawauchi, Y., J. Takasaki, and Y.M. Matsuura. 1994. Preparation and characterization of human rheumatoid arthritic synovial fluid phospholipase A2 produced by recombinant baculovirus-infected insect cells. J Biochem 116 82. 

Onishi, H., Maeda, K., Maeda, Y., Inoue, A., and Fujiwara, K. (1995). Functional chicken gizzard heavy meromyosin expression in and purification from baculovirus-infected insect cells. Proc. Natl. Acad. Sci. USA 92, 704-708. [Published erratum appears in Proc. Natl. Acad. Sci. USA 1995 92, 3076],... 

P-gp associated ATPase is vanadate sensitive. A membrane product prepared from baculovirus infected insect cells containing this activity is now commercially available from Gentest Corp. (Woburn, Massachusetts, U.S.). Substrates of P-gp, such as verapamil, have been demonstrated to stimulate this vanadate-sensitive membrane ATPase (123). By determination of inorganic phosphate liberated in the reaction containing a P-gp preparation and a test compound, in the presence and absence of vanadate, one can determine if the test compound is a substrate/inhibitor of P-gp (123,422). Any compound that binds to P-gp would stimulate the magnesium-dependent ATPase, and thus, this method cannot distinguish between a substrate and inhibitor of P-gp. 

Table 18.1 Substrate (autocamtide-2) and calmodulin activation kinetics in recombinant CaMKII holoenzymes purified from Baculovirus-infected insect cells...

Schlaeger EJ, Foggetta M, Vonach JM, Christensen K (1993), SF-1 a low cost culture medium for the production of recombinant proteins in baculovirus infected insect cells, Biotechnol. Tech. 7 183-188. 

The proteins to be included in VLPs should be those necessary to confer the desired degree of immunogenicity. As a consequence, VLPs are often composed of more than one protein. In order to use baculovirus-infected insect cells to produce VLPs it is necessary to predefine the proteins to be included, since the expression of these proteins could determine the stability (Hyatt et al., 1993) and location - intra- or extracellular - of the particle (French and Roy, 1990). 

Table 18.2 Virus-like particles (VLPs) produced in baculovirus-infected insect cells...

Maranga L, Cruz PE, Aunins JG, Carrondo MJT (2002a), Production of core and virus-like particles with baculovirus infected insect cells, Adv. Biochem. Eng. Biotechnol. 74 183-206. 

Microsomes from baculovirus-infected insect cells expressing CYP1A1, CYP1A2, CYP2A6, CYP2B6,... 

Xue, H.W., Pical, C., Brearley, C., Elge, S., and Muller-Rober, B., 1999, A plant 126-kDa phosphatidylinositol 4-kinase with a novel repeat structure. Cloning and functional expression in baculovirus-infected insect cells. J. Biol. Chem. 274 5738-5745. 

Strauss A, et al. Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J. Biomol. NMR 2005 31 343-349. 

Two other viral vectors are extensively used. Vaccinia virus, a large DNA-containing virus, replicates in the cytoplasm of mammalian cells, where it shuts dovm host-cell protein synthesis. Baculovirus infects insect cells, which can be conveniently cultured. Insect larvae infected with this virus can serve as efficient protein factories. Vectors based on these large-genome viruses have been engineered to express DNA inserts efficiently. 

Characterization of Truncated Kirsten-Ras Purified from Baculovirus Infected Insect Cells Indicates Heterogeneity due to N-terminal Processing and Nucleotide Dissociation... 

Cell pellets from 4L of baculovirus infected insect cells were resuspended into 25mM Tris-HCL, pH8.0, 5mM DTT, 250mM sucrose and protease inhibitor cocktail tablets (Boehringer Mannheim). Cells were homogenized and centrifuged at 38,000 X g for 20 min. The supernatant was ultracentrifuged at 100,000 X g for 2hrs, filtered, and loaded over a 21.5mm ID X 15 cm DEAF column... 

Figure 1. A, SDS-PAGE of K-ras produced in baculovirus infected insect cells. Protein samples were electrophoresed on a 4-20 % SDS gel under reducing conditions. 1 MW standards, 2 preparative DEAE pool, 3 purified Kirsten ras. B, Mass distribution of the purified K-ras reconstructed from the ESI-MS using PE-Sciex MacSpec software.

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