Construction of Recombinant Baculoviruses.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

A Novel Formulation of Asparaginase Encapsulated into Virus-like Particles of Brome Mosaic Virus: In Vitro and In Vivo Evidence.

The interest in plant-derived virus-like particles (pVLPs) for the design of a new generation of nanocarriers is based on their lack of infection for humans, their immunostimulatory properties to fight cancer cells, and their capability to contain and release cargo molecules. Asparaginase (ASNase) is an FDA-approved drug to treat acute lymphoblastic leukemia (LLA); however, it exhibits high immunogenicity which often leads to discontinuation of treatment. In previous work, we encapsulated ASNase into bacteriophage P22-based VLPs through genetic-directed design to form the ASNase-P22 nanobioreactors. In this work, a commercial ASNase was encapsulated into brome mosaic virus-like particles (BMV-VLPs) to form stable ASNase-BMV nanobioreactors. According to our results, we observed that ASNase-BMV nanobioreactors had similar cytotoxicity against MOLT-4 and Reh cells as the commercial drug. In vivo assays showed a higher specific anti-ASNase IgG response in BALB/c mice immunized with ASNase encapsulated into BMV-VLPs compared with those immunized with free ASNase. Nevertheless, we also detected a high and specific IgG response against BMV capsids on both ASNase-filled capsids (ASNase-BMV) and empty BMV capsids. Despite the fact that our in vivo studies showed that the BMV-VLPs stimulate the immune response either empty or with cargo proteins, the specific cytotoxicity against leukemic cells allows us to propose ASNase-BMV as a potential novel formulation for LLA treatment where in vitro and in vivo evidence of functionality is provided.

Immunogenicity and protective efficacy of yeast extracts containing rotavirus-like particles: a potential veterinary vaccine.

Rotavirus is the most common cause of severe diarrhea in many animal species of economic interest. A simple, safe and cost-effective vaccine is required for the control and prevention of rotavirus in animals. In this study, we evaluated the use of Saccharomyces cerevisiae extracts containing rotavirus-like particles (RLP) as a vaccine candidate in an adult mice model. Two doses of 1mg of yeast extract containing rotavirus proteins (between 0.3 and 3 µg) resulted in an immunological response capable of reducing the replication of rotavirus after infection. Viral shedding in all mice groups diminished in comparison with the control group when challenged with 100 50% diarrhea doses (DD50) of murine rotavirus strain EDIM. Interestingly, when immunizing intranasally protection against rotavirus infection was observed even when no increase in rotavirus-specific antibody titers was evident, suggesting that cellular responses were responsible of protection. Our results indicate that raw yeast extracts containing rotavirus proteins and RLP are a simple, cost-effective alternative for veterinary vaccines against rotavirus.