AbstractStrategies to augment immunity to self/neoantigens expressed by cancers are urgently needed to expand the proportion of patients benefiting from immunotherapy, particularly for GI cancers where only a fraction of patients respond to immunotherapies. However, current vaccine strategies are limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined a prime-boost strategy using a chimeric adenoviral vector (Ad5.F35) that resists pre-existing immunity followed by recombinant Listeria monocytogenes (Lm) to amplify immunity to the GI cancer antigen GUCY2C. This previously unexplored combination enhanced the quantity, avidity, polyfunctionality, and antitumor efficacy of GUCY2C-specific effector CD8⁺ T cells, without toxicity in any tissue, including GUCY2C-expressing intestines and brain. Importantly, this combination was partially resistant to pre-existing immunity to Ad5 which is endemic in human populations and vector-specific immunity did not limit the ability of multiple Lm administrations to repeatedly enhance GUCY2C-specific responses. Broadly, these findings suggest that cancer patient immunizations targeting self/neoantigens, as well as immunizations for difficult infectious diseases (HIV, malaria, etc), may be most successful using a combination of Ad5.F35-based priming, followed by Lm-based boosting. More specifically, Lm-GUCY2C may be utilized to amplify GUCY2C-specific immunity in patients receiving adenovirus-based GUCY2C vaccines currently in clinical trials to prevent or treat recurrent GI cancer.