Adenovirus Vaccine Expressing GUCY2C-S1

Adenovirus containing mouse extracellular domain (GUCY2C_1-429) with the influenza HA_107-119 CD4⁺ T-cell epitope known as site 1 (S1) was described previously (Ad5-GUCY2C-S1).20 (link) Here, GUCY2C-S1 was cloned into pShuttle and subcloned into the E1 region of previously generated replication-deficient chimeric adenovirus (Ad5.F35) in which the Ad5 fiber was replaced by the Ad35 fiber22 (link) to generate Ad5.F35-GUCY2C-S1. All adenovirus vaccines used in this study were produced in HEK293 cells and purified by cesium chloride ultracentrifugation under Good Laboratory Practices by the Baylor College of Medicine in the Cell and Gene Therapy Vector Development Lab and certified to be negative for replication-competent adenovirus, mycoplasma, and host cell DNA contamination. In vitro GUCY2C-expression experiments (dose–response and time–course) were carried out in A549 (American Type Culture Collection (ATCC)) cells. Virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points. Relative GUCY2C levels were quantified in supernatants by western blot using 2 μg/mL MS7 mouse anti-GUCY2C monoclonal antibody23–25 (link) and 0.1 μg/mL horseradish peroxidase-conjugated goat antimouse secondary antibody (Jackson Immuno).