Current influenza vaccine production procedures (use of embryonated chicken eggs) inherently limit the amount of influenza vaccine that can be produced prior to each year's flu season. Most of the influenza vaccine sold is inactivated. Inactivation of the virus is accomplished through the use of agents such as formalin which is a compound that is known to cross-link protein and damage epitopes. There is a clear need for new technologies that can be used to respond quickly to influenza outbreaks and pandemics and produce sufficient doses of high quality vaccine for the U.S. population. There is also need for improved vaccine formulations with increased immunogenicity and efficacy. The overall goal of this research proposal is to evaluate recombinant subunit proteins of the avian influenza strain A/Hong Kong/156/97 (H5N1) as vaccine candidates. Recombinant subunits of the HA, NP and M1 proteins will be expressed in the Drosophila S2 expression system. The recombinantly expressed HA, NP and M1 proteins will be highly purified by column chromatography. The quality of the Drosophila and Bomftyx-expressed proteins is anticipated to be superior to conventionally produced influenza proteins due to the fact that the protein will not be altered by formalin or other agents used for inactivation which can cross-link proteins and destroy epitopes. The subunit proteins will be purified and tested in different combinations as vaccine immunogens in a mouse immunogenicity model. The proteins will also be expressed in the Bombyx mori system to provide Hawaii Biotech Inc an alternative expression system to the Drosophila system. Humoral and cellular immune responses will be evaluated in the mouse model. Dose ranging studies and different adjuvants will also be evaluated. The Drosophila and Bombyx mori expressed HA proteins will be compared to commercially available baculovirus expressed HA protein in the mouse immunogenicity model. Specific optimized vaccine formulations will then be evaluated in a mouse challenge model. In addition, the role of NP and M1 proteins in cellular immune responses will be explored.