Methods of preparing carboxy-terminally truncated recombinant flavivirus

Abstract

The Flaviviridae comprise a number of medically important pathogens that cause significant morbidity in humans including the dengue (DEN) virus, Japanese encephalitis (JE) virus, tick-borne encephalitis virus (TBE), and yellow fever virus (YF). Flaviviruses are generally transmitted to vertebrates by chronically infected mosquito or tick vectors. The viral particle which is enveloped by host cell membranes, comprises a single positive strand genomic RNA and the structural capsid (CA), membrane (M), and envelope (E) proteins. The E and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E is glycosylated and contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. Problems have arisen in the art with respect to producing recombinant forms of the E glycoprotein that retain their native configuration and attendant properties associated therewith (i.e., ability to induce neutralizing antibody responses). To date, recombinantly produced E glycoproteins have suffered from a number of limitations including improper glycosylation, folding, and disulfide bond formation. The claimed invention has addressed these concerns by providing secreted recombinant forms of the E glycoprotein that are highly immunogenic and appear to retain their native configuration. Carboxy-terminally truncated forms of E containing the amino terminal 395 amino acids and a suitable secretion signal sequence were generated in Drosophila melanogaster Schneider cell lines. The recombinant proteins produced by this expression system should prove useful, inter alia, as immunogens and diagnostic reagents.