Development of Bluetongue Virus Multicomponent Vaccines Using a Novel Baculovirus System

The difficulties associated with obtaining completely safe, live virus vaccines by the traditional procedures of virus passage and selection of attenuated forms, as well as the risks and expenses involved in preparing killed or subunit vaccines from virulent viruses, may now be overcome using recombinant DNA technologies. Genetic engineering offers a variety of ways of preparing viral vaccines using expression vector systems. Using baculovirus multiple gene expression vectors based on Autographa cali/ornica nuclear polyhedrosis virus (AcNPV) has provided a new strategy for vaccine development. Such vectors are capable of the simultaneous ex pression of several foreign genes within the host insect cell in which AcNPV replicates. Using this technology, we have developed multicomponent virus-like particles (VLPs) as virus vaccines and used them as vaccine delivery systems for multiple immunogens. The VLP technology is a completely new technical development from a novel baculovirus expression vector system, which has the capability of simultaneously co-expressing up to five proteins in insect cell culture. The transformed cells are stable, and authentic protein product is produced at high levels. This novel technology has been used for the production of pilot-scale quantities of a recombinant bluetongue virus (BLU) multi-subunit vaccine (VLPs immunologically indistinguishable from BLU). Preliminary clinical trials have verified this vaccine's safety and efficacy. Unlike live virus vaccines, VLPs are non-infectious and lack virus (or other) DNA/RNA required for replication. VLPs do not replicate in host cells. However, trials in sheep have shown that VLPs are more immunogenic than subunit vaccines (viral proteins) or than viruses inactivated chemically. In addition, VLPs are effective at eliciting humoral, cell-mediated and mucosal immunities, and are safe to produce and handle. (As the baculovirus vector and host cells used to make VLPs do not come from mammalian sources, they do not contain mammalian derived pathogens.) The expression system described in this paper is a tool which may have a range of applications in industries employing biotechnology to produce vaccines, insecticides, or diagnostic and protein reagents. Diagnostic reagents developed for BLU and African horsesickness virus are discussed as examples. GENETIC engineering offers a variety of approaches for viral vaccines. An exciting advance in this field is the ability to construct virus-like particles (VLPs) which resemble their natural counterparts but lack genetic information (Le. are unable to replicate). We have developed VLPs based on bluetongue * NERC Institute of Virology & Environmental Microbiology, Oxford, U.K. (address for correspondence); Laboratory of Molecular Biophysics, University of Oxford, U.K.; and University of Alabama at Birmingham, USA. 183 virus (BLU), the causative agent of bluetongue disease of sheep. To date, 24 BLU serotypes (BLUI, BLU2, etc.) have been identified from different parts of the world (Erasmus 1990). In the past, live attenuated orbiviruses have been used as reasonably effective vaccines in those regions where bluetongue virus causes epidemics of disease in livestock. However, as the BLU genome comprises 10 double-stranded (ds) RNA species, there is some concern that using live attenuated viruses in vaccines may maintain the virus in the wild, and facilitate RNA segment reassortment and the generation of new viral genotypes. This is especially important as not all the attenuated derivatives of the known BLU serotypes appear adequately attenuated. The Bluetongue Virus The architecturally complex, 810 A diameter bluetongue virus can attach to, and replicate in, a variety of vertebrate cells (Roy, 1996). The virion contains seven structural proteins, four of which are organised into two protein shells. The outer shell contains two major protein species, VP2 (110 kD) and VP5 (58 kD), while the inner shell has two other major species, VP3 (100 kD) and VP7 (38 kD). The icosahedral inner shell encapsidates the virus genome and three other (minor) proteins; VPI (I50 kD), VP4 (76 kD) and VP6 (36 kD). In addition, three or four non-structural proteins are synthesised in virusinfected cells. Of the ten proteins, only VP2 and VP5 vary from serotype to serotype. All five core proteins and the three non-structural proteins are highly conserved (Roy et al. J990a). Each protein is encoded by a single BLU dsRNA segment (Mertens et al. 1984). To develop rationally-designed BLU vaccines in the past few years, we have been developing an understanding of the structural and functional relationships of BLU genes and gene products, and of the assembly of the gene products for the formation of virions. Baculovirus Expression Vectors Recent advances in gene manipulation have made it possible to express foreign genes in heterologous systems. The productivity and flexibility of insect baculovirus expression vectors, and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA in Spodoptera fru· giperda insect cells, have permitted this system to be used for the expression not only of a single gene, but also for the simultaneous expression of dual and multiple genes. To accomplish this, several expression vectors have been developed based on the resident promoters of AcNPV, the nuclear polyhedrosis virus of Autographa californica (B ishop 1992; Belyaev and Roy 1993; Belyaev et al. 1995). Using these various expression vectors, we have expressed all 10 BLU genes, either individually or in various combinations, using single, dual, triple and quadruple expression vectors, and have analysed the structure-function of each gene and gene product (Roy 1992). However, only the structural characteristics and functional attributes of those proteins relevant to generation of vaccines are discussed in this paper. 184 Assembling BLU Core-like Structures with Baculovirus Expression Systems