1088. Antigen Epitopes Expressed in Different Adenovirus Capsid Proteins Induce Different Levels of Epitope-Specific Immunity

Abstract

Top of pageAbstract The immune response against adenovirus gene transfer vectors (Ad) is directed against the capsid proteins and transgene expressed by the vector. Incorporation of pathogen-derived epitopes into capsid proteins may be a useful strategy to evoke an anti-pathogen immunity and may complement the anti-transgene immune response. Our previous data (Worgall et al Mol Ther 2004; 9:S261) demonstrate that incorporating an epitope of the outer membrane protein of Pseudomonas aeruginosa into the hexon of the adenovirus capsid provides protection against pulmonary challenge by P. aeruginosa in experimental animals. In this study, we compare the immune response against an epitope incorporated into different capsid proteins (hexon, fiber, penton base and protein IX) and determine if the immune response against the epitopes will be proportional to the number of copies of that protein in the capsid. To test this hypothesis, we used the major antigenic site HA from the hemagglutinin protein of influenza virus type A as model epitope. Replication deficient Ad vectors were engineered expressing the HA sequence YPYDVPDYAGG on the surface of the adenoviral virion as part of following 4 capsid proteins: in HVR5 loop of hexon protein (Ad.Hexon-HA), in HI loop of fiber protein (Ad.Fiber-HA), in RGD loop of penton protein (Ad.Penton-HA), and attached to the C-terminus of Protein IX (Ad.pIX-HA). The HA epitope is present at various copy numbers in each virion: hexon, 720; protein IX 240; penton base 60; and fiber 36. Using an anti-HA antibody, in vitro studies demonstrated that the modified capsid proteins were incorporated into virions and express the HA-sequences in the expected capsid proteins of each recombinant vector. CBA mice were immunized by the intramuscular route with the same dose of adenoviral vectors (5|[times]|109 particle units) and serum anti-HA titer was evaluated at 4 wk by ELISA against purified recombinant HIS-tagged HA fusion protein. Titers against HA in Ad.Fiber-HA immunized mice were three times higher compared to Ad.Hexon-HA immunized mice (555|[plusmn]|40 vs 162|[plusmn]|36, p IgG2b for Ad.Fiber-HA and Ad-Hexon-HA. In contrast to the variable response to a specific antigen inserted into different locations of the capsid, anti-Ad capsid titer were the same for all 4 HA modified vectors and also for an AdNull vector with the wild type capsid delivered by the same dose and route. We conclude that incorporation of an epitope into the fiber gene is the most potent strategy to elucidate an anti-epitope response, at least for this model system . The fact that immune response was independent of the dose of the epitope suggested that antigen position in the capsid and its recognition by B cells is the critical component of the response to the epitope incorporated into the Ad capsid.