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ABSTRACTSUMMARY We propose to continue our SBIR phase I work and further develop a broadly neutralizingpossibly universal influenza vaccine composed of virus like particlesVLPsdisplaying remodeled HA molecules which exhibit otherwise cryptic epitopesThese remodeled HAs will be expressed recombinantly in forms lacking or shedding the dominant hypervariable epitopes and instead display distinctly conserved subdominant antigenic sites known to promote an antibody response that will neutralize a broad spectrum of influenza virusesAdditionallythe VLP structure displaying the remodeled HA and NA contains more conserved influenza antigens like matrices MMand the nucleoprotein NPThe protective scope of current influenza vaccines is restricted to homologous viruses or closely related variants and vaccine efficacy wanes following the fast antigenic evolution of the influenza virusMost protective antibodies target highly variable and dominant sites on the globular head of the HA moleculealthough more conserved and less immune recognized conformational antigenic sites are also present in the stemHAand between the globular headHAand stem portions of HAIsolated human antibodies directed toward these sites have been found to neutralize a broad spectrum of influenza virusesIt seems reasonable therefore to prepare and test vaccines that display these highly conserved subdominant antigenic sites together with other important influenza antigens and determine if they stimulate a broad antibody response which is minimal in a natural influenza infection or following vaccination with formulations containing whole HA moleculesIncorporation of remodeled HA molecules into influenza virus like particlesVLPsshould provide an excellent opportunity to develop a broadly neutralizing vaccineVLPs are generated by the co expression of five structural influenza proteinsMMHANA and NPand do not contain infectious viral genetic material and are therefore unable to replicate or cause infectionStudies performed during the phase I SBIR showed that VLP generated with remodeled HA not only elicit neutralizing antibodies but also afford significant protection against a lethal influenza challenge after a single immunizationTo continue this workwe propose to further evaluate the VLP based vaccine containing alternative conformations of the remodeled HA together with native or modified NA as surface component of a MMand NP influenza virus like particle assemblyThis vaccine is produced in suspension culture of mammalian cells suitable for the most advanced fermentation technology and amenable for stable cells line development for continuous manufacturingThis vaccine composition and configuration will elicit humoral neutralizing immunity as well as cellmediated immunityCMItoward the Mand NP antigensThe immunogenicity and efficacy of the VLP vaccine compositions will be tested in mice and ferrets using as challenge influenza virus subtypes that represent groupand groupInfluenza A virusA similar strategy will be pursued with the two lineages of influenza B virusesThese studies will provide the foundation to advance development of an influenza vaccine able not only to broaden and enhanced the spectrum of protection but also extend the duration on immunity for several yearsInfluenza is a constant threat and the current vaccine technology to prevent seasonal or potential pandemic outbreaks is in a permanent catch up mode not only to produce vaccine on time but also to correctly match the antigenic composition of prevailing circulating virusEven in the best of circumstances the overall effectiveness of the influenza vaccine may range fromtoTo change this paradigmwe need a vaccine technology that overcomes and withstands antigenic variation and prolongs the duration of induced immunity as well as utilizes rapidefficient and cost effective vaccine manufacturing methodsThis proposal addresses these issues aiming to advance the development of the vaccine technology and production methods to overcome the limitation of the current vaccine and its production NARRATIVE Development of a broadly protectiveuniversalvaccine able to withstand antigenic variation and sustain efficacy for an extended time should have a major impact on influenza prevention and controlTo achieve this goalwe propose to create influenza virus like particlesVLPsdisplaying remodeled HA molecules revealing distinctly conserved subdominant antigenic sites known to elicit an antibody response that will neutralize a broad spectrum of influenza virusesPreliminary studies have shown promising results thus we describe plans to continue work on developing not only a more effective vaccine but also a rapidflexible and cost effective manufacturing system