Despite the encouraging development of a preventive vaccine for human papillomavirus (HPV), it cannot improve ongoing infections. Therefore, a new vaccine is urgently needed that can prevent and treat cervical cancer, and cure pre-cancerous lesions. In this study, we constructed two peptide-based vaccines. The first was a short-term, long-peptide (ST-LP) vaccine that simultaneously targeted three key carcinogenic epitopes (E5-E6-E7) on HPV16. We tested this vaccine in murine TC-1 cells infected with a recombinant adeno-associated virus (rAAV) fused with HPV16E5 DNA (rTC-1 cells), which served as a cell model; we also tested it in immune-competent mice loaded with rTC-1 cells, which served as an ectopic tumor model. The ST-LP injections resulted in strong, cell-mediated immunity, capable of attacking and eliminating abnormal antigen-bearing cells. Furthermore, to prolong immunogenic capability, we designed a unique rAAV that encoded the three predicted epitopes for a second, long-term, long-peptide (LT-LP) vaccine. Moreover, we used a new immune strategy of continuous re-injections, where three ST-LP injections were performed at one-week intervals (days 0, 7, 14), then one LT-LP injection was performed on day 120. Our in vitro and in vivo studies revealed that this strategy could boost the immune response to produce longer and stronger protection against target cells, and mice were thoroughly protected from tumor growth. Our results showed that priming the immune system with the ST-LP vaccine, followed by boosting the immune system with the LT-LP vaccine could generate a rapid, robust, durable cytotoxic T-lymphocyte response to HPV16-positive tumors. (C) 2015 Elsevier Ltd. All rights reserved.