Dityrosine Locked Prefusion F Protein: A Path To A Protective RSV Vaccine

DESCRIPTION provided by applicant The pre fusion conformation of the RSV F protein preF is the primary determinant of neutralization in immunized sera However this conformation is unstable and rapidly transitions to the postfusion conformation A disulfide stabilized prefusion variant with cavity filling mutations DS Cav has been shown to yield high neutralizing titers in animals however it only transiently maintains its native prefusion conformation Avatar Medical LLC has developed a proprietary technology that locks immunogens in native conformations that better present broadly protective epitopes Locking is accomplished by introducing targeted dityrosine DT crosslinks into the fully folded native protein We will apply this technology to lock the F protein fully in its pre fusion conformation DT preF in order to develop a stable recombinant preF based RSV vaccine immunogen Our minimally modifying conformational locking technology enzymatically introduces zero length covalent bonds into proteins and complexes after the protein has fully folded Bonds only form between Tyr side chains in close structural proximity and thus lock proteins in their native conformation while preserving structural integrity DT locking involves steps i expressing and purifying soluble F proteins with targeted conservative to Tyr substitutions and ii enzymatically crosslinking the complex in its pre fusion conformation Our DT locked preF immunogens will focus immune responses on potently neutralizing epitopes that are only displayed in the preF conformation and away from non or weakly neutralizing postfusion epitopes As a result vaccination with DT preF should elicit potent and lasting protection against RSV In this Phase I application we propose to design and characterize DT preF variants and confirm that they maintain the native prefusion conformation using a panel of preF specific mAbs We will then demonstrate that our DT preF immunogen elicits higher neutralizing titers in mice compared to DS Cav In Phase II we will perform pre clinical testing in cotton rat challenge studies for efficacy and in rabbits for safety with a view toward filing an IND with th FDA To accomplish the Phase I goals we will carry out the following Specific Aims I Design DT preF variants in the WT disulfide and DS Cav stabilized backgrounds and characterize them antigenically and biochemically by comparison to uncrosslinked and DS Cav controls Milestone Selection of three DT locked F proteins that retain binding to key preF specific nAbs II Perform immunogenicity studies in mice and determine the neutralization titers induced by our DT preF variants compared to DS Cav as well as postF Formalin inactivated RSV and live RSV virus controls Milestone Immunization with DT preF elicits higher neutralization titers than DS Cav PUBLIC HEALTH RELEVANCE There is currently no effective vaccine for RSV and each year RSV infects million children in the US and is the leading cause of infant hospitalizations Globally it accounts for of deaths in infants second only to malaria and in addition it poses a serious threat to the elderly and immuno compromised We propose to apply our proprietary protein engineering approach to the design of an RSV vaccine immunogen based on recombinant F fusion protein stabilized in its pre fusion conformation that will trigger the production of antibodies in vaccinated individuals that will bind to and neutralie the virus when it enters the body