PURPOSE: Human papillomavirus (HPV) infection is the major cause of (pre)malignant cervical lesions. We previously demonstrated that Vvax001, a replication-incompetent Semliki Forest virus vaccine encoding HPV type 16 (HPV16) E6 and E7, induced potent anti-E6 and -E7 cytotoxic T-cell responses. In this study, we investigated the clinical efficacy of Vvax001 in patients with HPV16-positive cervical intraepithelial neoplasia (CIN) grade 3 (CIN3). PATIENTS AND METHODS: Patients with newly diagnosed HPV16-positive CIN3 were eligible for participation. Patients received three immunizations of Vvax001 (5 × 107 infectious particles) at a 3-week interval. Up to 19 weeks after the last immunization, patients were monitored for regression of CIN3 by colposcopy. A colposcopy-guided biopsy was taken at the last visit, and a standard-of-care loop excision was performed only in case of remaining CIN grade 2/CIN3. Histopathologic response rates, HPV16 clearance, treatment-related adverse events, and vaccine-induced immune responses were assessed. RESULTS: A total of 18 patients were enrolled and fully immunized. Colposcopic examination revealed a reduction in CIN3 lesion sizes in 17/18 (94%) patients already evident from 3 weeks onward after the last immunization. A histopathologic complete response (regression to CIN grade 1 or no dysplasia) was observed in 9/18 patients (50%) and HPV16 clearance in 10/16 patients (63%). Vvax001 did not induce clearance of other HPV types. To date, no recurrences have been observed, with a median and longest disease-free survival of 20 and 30 months, respectively. No serious adverse events were observed. CONCLUSIONS: Treatment with Vvax001 is safe and feasible and shows preliminary clinical effectiveness in patients with HPV16-associated CIN3 lesions.