PROJECT SUMMARY It is estimated that human papillomavirus (HPV) is responsible for 42,700 cancers in the US each year and that it caused more than 90% of anal and cervical cancers and about 70% of oropharyngeal, vaginal, and vulvar cancers. The increases in incidences of HPV-associated anal and oropharyngeal cancers in the US are notable, and cervical cancer is the fourth most common cancer among women globally. This is the second competitive renewal of this R01, which has the long-term goal of developing therapeutic vaccines for HPV. This is significant because currently available prophylactic vaccines are effective only for preventing HPV infections, but not for eliminating them once they are established. The estimated lifetime probably of acquiring HPV is 91.3% for men and 84.6% for women. We developed and are testing PepCan, a therapeutic vaccine that consists of four “current good manufacturing grade” synthetic peptides that cover the E6 protein of HPV type 16 (HPV 16), along with a Candida skin-test reagent as a novel vaccine adjuvant. Our dose-escalation single- site Phase I clinical trial of PepCan to treat women with cervical high-grade squamous intraepithelial lesions (HSILs) was recently completed. It demonstrated safety, a significant decrease in HPV 16 viral load, and a significant increase in circulating T-helper type 1 cells after vaccination. A randomized (to two treatment arms), double-blind, Phase II clinical trial is on track to complete recruitment soon. Furthermore, in a double-blind, placebo-controlled (3:1 ratio) Phase I/II clinical trial, PepCan is being tested to reduce recurrence of squamous cell carcinoma of head and neck in patients who reach “no evidence of disease” status after curative treatment. The following aims will test our hypothesis that PepCan induces HPV-specific immune responses, resulting in reduced recurrence of head and neck cancer (HNC) and increased regression of HSILs when the effector cells (i.e., T-cells) reach the disease sites. Aim 1: Assess efficacy and safety of our HPV therapeutic vaccine for HNC recurrence reduction and cervical HSIL regression Aim 2: Examine immune factors that influence PepCan response and vaccine mechanisms Aim 3: Examine how microbiomes of gut and of disease sites (cervix and mouth) contribute to vaccine efficacy Results of the proposed investigations may lead to FDA approval of a therapeutic vaccine for two HPV-related cancers and to an improved understanding of immunotherapy mechanisms.