Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as x-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX® adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX® adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.

While several West Nile vaccines are being developed, none are yet available for humans. In this study aimed at developing a vaccine for humans, West Nile virus (WNV) envelope protein (E) and non-structural protein 1 (NS1) were produced in the Drosophila S2 cell expression system. The Cterminal 20% of the E protein, which contains the membrane anchor portion, was deleted, thus allowing for efficient secretion of the truncated protein (80E) into the cell culture medium. The proteins were purified by immunoaffinity chromatography (IAC) using monoclonal antibodies that were flavivirus envelope protein group specific (for the 80E) or flavivirus NS1 group specific (for NS1). The purified proteins were produced in high yield and used in conjunction with adjuvant formulations to vaccinate mice. The mice were tested for both humoral and cellular immune responses by a plaque reduction neutralization test and ELISA, and by lymphocyte proliferation and cytokine production assays, respectively. The results revealed that the 80E and the NS1 proteins induced both high-titered ELISA and neutralizing antibodies in mice. Splenocytes from immunized mice, cultured in vitro with the vaccine antigens as stimulants, showed excellent proliferation and production of cytokines . The level of antigen-stimulated lymphocyte proliferation and cytokine production was comparable to the level obtained from mitogen (phytohemagglutinin or pokeweed) stimulation, indicating a robust cellular response as well. These findings are encouraging and warrant further in vivo studies to determine the protective efficacy of the WNV vaccine candidate.

Two mannose-negative mutants of Escherichia coli K-12 have been isolated. These mutants are deficient in the ability to synthesize phosphomannose isomerase and capsular polysaccharide when grown on glucose-containing media. Interrupted mating experiments to determine the kinetics of genetic transfer show that the two mannose-negative mutations map together between the histidine and tryptophan regions of the E. coli chromosome. The capsular polysaccharide of Escherichia coli K-12 contains glucuronic acid, D-galactose, Dglucose, and L-fucose in the approximate molar

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 g of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzymelinked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 g of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1-or 25-g dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.

Infections with filoviruses in humans are highly virulent, causing hemorrhagic fevers which result in up to 90% mortality. In addition to natural infections, the ability to use these viruses as bioterrorist weapons is of significant concern. Currently, there are no licensed vaccines or therapeutics available to combat these infections. The pathogenesis of disease involves the dysregulation of the host's immune system, which results in impairment of the innate and adaptive immune responses, with subsequent development of lymphopenia, thrombocytopenia, hemorrhage, and death. Questions remain with regard to the few survivors of infection, who manage to mount an effective adaptive immune response. These questions concern the humoral and cellular components of this response, and whether such a response can be elicited by an appropriate prophylactic vaccine. The data reported herein describe the production and evaluation of a recombinant subunit Ebola virus vaccine candidate consisting of insect cell expressed Zaire ebolavirus (EBOV) surface glycoprotein (GP) and the matrix proteins VP24 and VP40. The recombinant subunit proteins are shown to be highly immunogenic in mice, yielding both humoral and cellular responses, as well as highly efficacious, providing up to 100% protection against a lethal challenge with live virus. These results demonstrate proof of concept for such a recombinant non-replicating vaccine candidate in the mouse model of EBOV which helps to elucidate immune correlates of protection and warrants further development.