Protein Production with Recombinant Baculoviruses in Lepidopteran Larvae.

With an increasing need for functional analysis of proteins, there is a growing demand for fast and cost-effective production of biologically active eukaryotic proteins. The baculovirus expression vector system (BEVS) is widely used, and in the vast majority of cases cultured insect cells have been the host of choice. A low cost alternative to bioreactor-based protein production exists in the use of live insect larvae as "mini bioreactors." In this chapter we focus on Trichoplusia ni as the host insect for recombinant protein production, and explore three different methods of virus administration to the larvae. The first method is labor-intensive, as extracellular virus is injected into each larva, whereas the second lends itself to infection of large numbers of larvae via oral inoculation. While these first two methods require cultured insect cells for the generation of recombinant virus, the third relies on transfection of larvae with recombinant viral DNA and does not require cultured insect cells as an intermediate stage. We suggest that small- to mid-scale recombinant protein production (mg-g level) can be achieved in T. ni larvae with relative ease.

Baculovirus Insecticide Production in Insect Larvae.

Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field tests with such recombinant baculoviruses have been done in the past, and more tests are ongoing. However, low-cost production of recombinant baculovirus in larvae poses specific problems, due to the short survival time of the production host.In this chapter, benchtop-scale production of two typical baculoviruses is described. First, we describe the production of wild type Helicoverpa zea nucleopolyhedrovirus in bollworm (H. zea) larvae. H. zea larvae are very aggressive and need to be reared in isolation from each other. Second, we describe the production of a recombinant Autographa californica multiple nucleopolyhedrovirus in the non-cannibalistic cabbage looper, Trichoplusia ni. The recombinant baculovirus encodes the insect-specific scorpion toxin LqhIT2. The tetracycline transactivator system enables the production of wild-type quantity and quality product while toxin expression is repressed since normal toxin production would result in premature death of the production host that would limit progeny virus production.

Recombinant protein production in insect larvae: host choice, tissue distribution, and heterologous gene instability.

The expression of the fluorescent protein, DsRed, facilitates the optimization of protein production in orally-infected whole larvae. Trichoplusia ni was shown to be a much better host for recombinant AcMNPV compared to four other noctuid Lepidopteran species achieving 100% infectivity at the minimal tested dose. The highest density of marker protein was found in endothelial and tracheal cells, fat body, and hemocytes. Trichoplusia ni larvae possessed visually detected color over sequential passages of oral infection until the sixth round. Western blot analysis confirmed the progressive decrease of both tetramer and monomer forms of DsRed. The intact DsRed gene and promoter region was present in late passages, but viral population carrying the heterologous gene had dropped more than 2-logs after the fifth round while the amount of total viral DNA remained unchanged over sequential passages.

Baculovirus insecticide production in insect larvae.

Baculovirus-based insecticides are currently being used worldwide, and new products are in development in many countries. The most dramatic examples of successful baculovirus insecticides are found in soybean in Brazil and cotton in China. Production of baculoviruses is generally done in larvae of a convenient host species, and the level of sophistication varies tremendously between field-collection of infected insects at the one extreme and automated mass manufacturing at the other. Currently, only products with wild type baculoviruses as active ingredients are commercially available. Baculoviruses encoding insecticidal proteins are considered attractive, especially for crops with little tolerance to feeding damage, where speed-of-kill is an important characteristic. Successful field tests with such recombinant baculoviruses have been done in the past, and more tests are ongoing. However, low-cost production of recombinant baculovirus in vivo poses specific problems, because of the short survival time of the production host. In this chapter, benchtop-scale production of two typical baculoviruses is described. First, the authors describe the production of wild type Helicoverpa zea nucleopolyhedrovirus in larvae of the bollworm, H. zea. Larvae of this species are very aggressive and need to be reared in isolation from each other. The authors then describe the production of a recombinant Autographa californica multiple nucleopolyhedrovirus in the noncannibalistic cabbage looper, Trichoplusia ni. The recombinant baculovirus encodes the insect-specific scorpion toxin LqhIT2, which severely limits progeny virus production. The tetracycline transactivator system enables the production of wild-type quantity and quality product while toxin expression is repressed.

Production of a recombinant antibody fragment in whole insect larvae.

Infection of insect cells with baculovirus expression constructs is commonly used to produce recombinant proteins that require post-translational modifications for their activity, such as mammalian proteins. However, technical restraints limit the capacity of insect cell-based culture systems to be scaled up to produce the large amounts of recombinant protein required for human pharmaceuticals. In this study, we designed an automated insect rearing system and whole insect baculovirus expression system (PERLXpress) for the expression and purification of recombinant proteins on a large scale. As a test model, we produced a recombinant mouse anti-botulinum antibody fragment (Fab) in Trichoplusia ni larvae. A recombinant baculovirus co-expressing the Fab heavy and light chains together with N-terminal sequences from the silkworm hormone bombyxin, to direct proteins into the secretory pathway, was constructed. Fifth instar larvae were reared and infected orally with recombinant (pre- occluded) baculovirus using the automated system and harvested approximately after 4 days. The total yield of recombinant Fab was 1.1 g/kg of larvae, resulting in 127 mg of pure Fab in one production run. The Fab was purified to homogeneity using immobilized metal affinity chromatography, gel filtration, and anion exchange chromatography. The identity of the purified protein was verified by Western blots and size-exclusion chromatography. Purified recombinant Fab was used to detect botulinum toxin in ELISA experiments, demonstrating that the heavy and light chains were properly assembled and folded into functional heterodimers. We believe that this is the first demonstration of the expression of a recombinant antibody in whole insect larvae. Our results demonstrate that a baculovirus-whole larvae expression system can be used to express functionally active recombinant Fab fragments. As the PERLXpress system is an automated and linearly scalable technology, it represents an attractive alternative to insect cell culture for the production of large amounts of human pharmaceuticals.