Using baculovirus expression vectors to produce virus-like particles (VLPs) is not new. Intact, non-infectious poliovirus1 and bluetongue virus2 VLPs were synthesised using some of the earliest baculovirus vectors with considerable success. In many of the projects we undertake for customers we have also generated VLPs for a wide range of different viruses. They are ideal candidates for use as sub unit vaccines. The immunogenic parts of the virus can be replicated without assembling an infectious entity. Although we can’t give specific details of these projects owing to client confidentiality we can offer some general guidelines about how best to produce different types of VLPs, based on their predicted structures.
It should be no surprise that baculovirus vectors are very good at producing VLPs. After all, they have to make their own infectious particles in the normal course of virus replication. This occurs before the very late phase of baculovirus gene expression when most recombinant proteins are made. If you want to make VLPs using baculovirus vectors one of the first things you should do is consider the nature of your recombinant target. If it is an enveloped VLP, such as one derived from influenza virus, you should use a parental virus vector that is optimal for producing membrane-bound or secreted proteins. This will maximise transport of the recombinant glycoproteins (e.g. haemagglutinin and neuraminidase) to the plasma membrane where it should be joined by the influenza virus matrix protein to promote budding of VLPs. Your recombinant virus should ideally incorporate copies of all three genes so that a single virus can be used to infect cells. If you have each gene in a different virus you can perform triple virus infection of insect cells but it will probably be less efficient. The ideal parental virus vector for making enveloped VLPs is our flashBAC™ ULTRA variant, which lacks several genes encoding proteins detrimental to the production of plasma membrane-bound or secreted glycoproteins.
Conversely, for some VLPs, which remain intracellular after their synthesis, it can be better to use a parental virus that retains the genes removed from flashBAC™ ULTRA. Two of these genes, p10 and cathepsin, encode proteins that promote virus-infected cell lysis in the very late stages of replication. We have noted that purification of certain VLPs, mainly those comprising small, protein-only components that are protease-resistant, is rendered easier if we use flashBAC™ PRIME. The VLPs preparations are much cleaner, being largely devoid of contaminating cellular material. They purify very nicely on either sucrose or caesium chloride gradients. This is particularly true if you are using cell lines derived from Trichoplusia ni. These cells seem to fall apart more readily in the latter stages of infection than those isolated from Spodoptera frugiperda (We will address the key differences between these two species in a later blog). While lysis of virus-infected cells may be counter intuitive for the production of recombinant proteins, it has certainly helped us to purify a number of difficult to produce VLPs.
If you are unsure about the best parental virus vector to use, why not take advantage of our flashBAC™ selection box. We can configure this according to your requirements and budget with a number of different flashBAC™ variants so that you can generate recombinant viruses and test expression simultaneously.
References
Urakawa, T.,Ferguson, M., Minor, P.D. Cooper, J., Sullivan, M., Jeffrey W. Almond, J.W. and Bishop, D.H.L (1989). Synthesis of Immunogenic, but Non-infectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector. Gen. Virol. 70, 1453-1463.
Loudon, P.T., Hirasawaa, T., Oldfield, S., Murphy, M. and Roy, P. (1991). Expression of the outer capsid protein vp5 of two bluetongue viruses, and synthesis of chimeric double-Shelled virus-like particles using combinations of recombinant baculoviruses. Virology 182, 793-801.