Background: With the increasing prevalence of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection, effective treatment strategies for HPV-positive HNSCC are urgently needed. Here, we present the results of neoadjuvant checkpoint blockade of pembrolizumab, therapeutic HPV DNA vaccine of GX-188E, and long-acting interleukin-7 of GX-I7 for patients with resectable HPV-16 and/or 18-positive HNSCC. Methods: In this single-arm, phase 2 trial, patients with resectable HPV-16 and/or 18-positive HNSCC were enrolled. Patients were given pembrolizumab 200mg on day 1 and day 22; GX-188E 2mg on day 1, 8, and 22; and GX-I7 1200 ug/kg on day 8 before surgical resection. Major pathologic response (MPR; defined as residual viable tumor of less than or equal to 10%) was primary endpoint with null and alternative hypothesis of MPR rate ≤5% and ≥35%, respectively. Secondary objectives were safety, recurrence, and survival. Results: Of the 11 patients included, all underwent surgical resection after neoadjuvant treatment without delays in surgery or increased surgical complications. MPR was achieved in seven patients (63.6%) and pathologic complete response was achieved in four patients (36.3%), which met the primary endpoint. Single cell RNA sequencing of baseline and post-treatment paired specimens revealed that the proportion of follicular helper T cell cluster, featured with CXCR5 and BCL6 expression, was significantly increased among tumor-infiltrating immune cells, accompanied with the reinvigoration of CD8 T cell cluster toward less exhausted phenotypes, represented with the upregulation of TCF7 and CD28 expression. In addition, artificial intelligence-powered spatial analysis revealed that triple combination significantly increased the density of tumor-infiltrating lymphocytes, completely converting immune-desert and immune-excluded type of tumor to inflamed immune phenotype. Conclusions: Neoadjuvant pembrolizumab, GX-188E, and GX-I7 showed promising activity and manageable safety profile in patients with resectable HPV-16 and/or 18-positive HNSCC. Therapeutic induction of brisk immune responses significantly reshaped the tumor microenvironment and associated with pathologic regression, warranting further investigation of pembrolizumab, GX-188E, and GX-I7 for patients with HPV-positive HNSCC. Clinical trial information: NCT05286060.