A Universal Flu Vaccine Based on Conformationally Locked Soluble Headless HA

DESCRIPTION provided by applicant An influenza pandemic remains an acute threat to world health and stockpiling a universally protective influenza vaccine provides a strong defense against this potential catastrophe The hemagglutinin HA protein is the primary target of humoral Ab responses to Influenza Vaccines based on full length HA give rise predominantly to strain specific Ab responses to the immunodominant and highly variable head domain and lack breadth of protection However the majority of the broadly neutralizing antibodies bnAbs that have been isolated to date target HAandapos s highly conserved stalk domain where they recognize trimer specific quaternary neutralizing epitopes QNEs An HA immunogen from which the head domain has been removed is expected to focus immune responses on these conserved stalk QNEs resulting in broadly protective Ab responses But current andquot Headless HAandquot constructs do not fold correctly or display only part of the stalkandapos s key QNE s and thus have only shown limited heterologous protection Avatar has developed a method of locking protein immunogens in their native quaternary conformation in order to better present broadly protective QNEs Targeted dityrosine DT crosslinks are engineered into fully folded native proteins by i introducing conservative Tyr substitutions and then ii catalyzing DT bond formation in order to conformationally lock the proteins DT bonds are catalyzed enzymatically and only form between Tyr side chains in very close structural proximity and unlike engineered disulfide bonds are introduced after the protein has fully folded DT bonds are safe and irreversible and because the bonds are zero length this approach fully preserves protein structure and furthermore avoids aggregation because DT bonds do not form spontaneously We will engineer DT bonds in the stalk of the intact HA trimer to lock it in its native conformatio This will allow us to subsequently remove the head by proteolysis while maintaining the stalkandapos s native trimeric structure generating a conformationally locked andquot DT Headless HAandquot To this end we will introduce both Tyr substitutions at targeted positions into the stalk and proteolytic cleavage sites into the head domain of the same HA molecule Following crosslinking of the stalk and proteolytic removal of the head we will analyze the DT locked stalk trimer antigenically by comparison to uncrosslinked and wild type controls AIM Then we will perform lethal viral challenge studies in mice to confirm that conformationally locked DT Headless HA elicits robust homologous protection and improved heterospecific neutralization responses compared to full length WT HA AIM By focusing Ab responses on the conserved stalk QNEs and away from the immunodominant head of HA the DT Headless HA immunogen is expected to give rise to broadly neutralizing Abs that protect from homologous and drift variants as well as group heterologous and perhaps even group heterologous challenges PUBLIC HEALTH RELEVANCE Influenza pandemic outbreaks remain an acute threat to world health and stockpiling a universally protective influenza vaccine would defend against such a potential catastrophe We propose to apply our protein engineering approach to the design of a universal influenza vaccine immunogen that will protect against all strains of the influenza virus including pandemic strains influenza by specifically triggering the production of antibodies in vaccinated individuals that will bind to and neutralize the virus when it enters the body