The use of subunit proteins in dengue immunoprophylaxis offers a number of theoretical advantages, but this strategy has not been adequately explored. The baculovirus-insect cell system has been widely used to produce protein products of foreign genes, because: (1) a high level of expression of the foreign gene can be achieved, and (2) in a number of instances the insect cell system can process and modify expressed products to yield functionally and immunologically active proteins. Recent studies of vaccinia virus expressed dengue virus envelope protein (E) showed that a C-terminally truncated E, approximately 80% in length, is more immunogenic and protective than the full-length E in mice. The increase in immunogenicity and efficacy associated with the specific truncation of E has implications for the development of effective subunit vaccines for dengue as well as other flavivirus diseases. For these reasons, we constructed a total of 8 recombinant baculoviruses that expressed 80% E or full-length E of 3 different dengue virus serotypes (type 4, 2, and 3) and Japanese B encephalitis (JE) virus. The baculovirus-expressed 80% E proteins of dengue type 4,2,3 and JE viruses was secreted into the medium fluid of the infected cells and was stable. Most animals immunized with the intracellular form of 80% E or the control 93% E developed signs of encephalitis but none succumbed to the lethal challenge. On the other hand, none of the mice immunized with extracellular forms of 80% E showed encephalitic symptoms following dengue virus challenge suggesting that these animals developed a higher level of resistance. Analysis of sera from immunized mice confirmed that extracellular 80% e induced a 3- to 5- fold higher antibody response than that of intracellular 80% E of 93% E. Similar results were obtained in the case of dengue type 2 and 3. Immunization of rhesus monkeys with these E proteins expressed by recombinant baculoviruses is in progress.
